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Patient A 33-year-old man diagnosed with extranodal natural killer cell/T-cell lymphoma, nasal type (ENKTCL-NT) inducing palatal perforation was referred to the perioperative oral care support center of Tohoku University Hospital for dental care to support cancer treatment including chemotherapy and hematopoietic stem cell transplantation (HSCT). Dental review during chemotherapy revealed mucositis suspected to be caused by mucosal trauma from altered jaw function (chewing and speech) due to palatal perforation. Although the patient was already in the cleanroom, an oral appliance as well as conservative care as recommended in oral management guidelines for HSCT were used to prevent worsening of oral mucositis at subsequent HSCT including High-dose chemotherapy and total body irradiation. After HSCT, a prosthodontist fitted a palatal obturator made by a dental technician and an oral surgeon reviewed the necrotic bone and removed the sequestra according to the changes in the palate. This approach involving a multidisciplinary team including a hematologist improved the impaired oral function and minimized oral complications . Discussion ENKTCL-NT and its treatment have a significant impact on patients' oral status. Hence, it is important to provide customized dental care based on previously endorsed guidelines according to the type of disease, treatment requirements, and oral and systemic status. Conclusions This report indicated the importance of dental care with a customized plan before, during, and after HSCT for ENKTCL-NT with multidisciplinary supportive care for cancer patients to improve the impaired oral function and to minimize oral complications.

One of the most severe complications of lung resection is postoperative pneumonia, and its prevention and prediction are critical. Exhaled acetone and isoprene are thought to be related to metabolism; however, little is known on their relationship with bacteria living in the oral cavity or their meaning in the acute phase in perioperative lung cancer patients. We measured acetone and isoprene in exhaled breath of 13 Japanese patients with lung cancer (3 women and 10 men, age range 62-82 years, mean 72.4 years) before breakfast during hospitalization, and compared with two acute-phase proteins, C-reactive protein (CRP) and albumin in blood serum, as well as the total number of bacteria in saliva and their activity to produce acetone and isoprene. Before operation, intensive oral care was carried out for each patient to prevent postoperative pneumonia, and swallowing and cough reflexes were measured for 12 of 13 patients to assess risk of postoperative pneumonia. Breath and saliva were sampled before intensive oral care (T1), after oral care but before operation (T2), and after operation (T3) during hospitalization. The total number of oral bacteria in saliva decreased significantly from T1 to T2 among 13 patients. No acetone or isoprene was detected from saliva after in vitro incubation under anaerobic or aerobic conditions, but both acetone and isoprene were detected in breath. After operation, breath acetone correlated significantly with CRP (Spearman's ρ = 0.559, P = 0.03), but not with albumin. Breath isoprene correlated significantly with albumin (Spearman's ρ = 0.659, P = 0.008), but not with CRP after operation. Although the number of subjects was small, our results support the hypothesis that breath acetone and isoprene may be related with these acute-phase proteins, which reflect inflammatory reactions and subsequent changes in metabolism in the early postoperative phase of lung resection.

Oral bacteria may contribute to postoperative infectious complications including postoperative pneumonia or surgical site infection. The aim of this study was to investigate the impact of preoperative dental care on postoperative outcomes among surgical patients under general anesthesia.Retrospective cohort study.We analyzed clinical records of major surgical patients at a university hospital between 2016 and 2018. Subjects were categorized into either the preoperative dental care group, those being referred to dentists by their surgeons based on an individual surgeon's judgment for dental care before surgery, or the control group.The primary outcome was postoperative infectious complications. Secondary outcomes were postoperative inflammation markers (C-reactive protein and fever), and economic outcomes (postoperative length of hospital stay and medical expenses). As the main analysis, the average treatment effects of the preoperative dental care were obtained from the augmented inverse-probability weighting (AIPW) method with consideration of demographics and perioperative risk factors to estimate causal effect of the intervention from the observational data. Then, stratified analyses by age and surgical sites were conducted with the inverse-probability weighting and linear regression methods, respectively.In the AIPW estimation, compared with the control group, the care group saw a significantly lower rate of postoperative infection (average treatment effect -3.02) and shorter fever duration (-2.79 days). The stratified analysis by age revealed significant positive impact of dental care in all age groups, including the highest treatment effects observed among patients younger than 60. Also, treatment effect was observed in wider surgical sites than previously known.This study indicates a significant impact of preoperative dental care on preventing postoperative infection and inflammation. Along with old age or certain types of surgeries in which advantages of dental referral have been already known, preoperative dental referral could be beneficial for broader types of patients.

Metabolites are sensitive indicators of moment-to-moment cellular status and activity. Expecting that tissue-specific metabolic signatures unveil a unique function of the tissue, we examined metabolomes of mouse liver and testis and found that an unusual metabolite, 2-hydroxyglutarate (2-HG), was abundantly accumulated in the testis. 2-HG can exist as D- or L-enantiomer, and both enantiomers interfere with the activities of 2-oxoglutarate (2-OG)-dependent dioxygenases, such as the Jumonji family of histone demethylases. Whereas D-2-HG is produced by oncogenic mutants of isocitrate dehydrogenases (IDH) and known as an oncometabolite, L-2-HG was the major enantiomer detected in the testis, suggesting that a distinct mechanism underlies the testicular production of this metabolite. We clarified that lactate dehydrogenase C (LDHC), a testis-specific lactate dehydrogenase, is responsible for L-2-HG accumulation by generating and analysing Ldhc-deficient mice. Although the inhibitory effects of 2-HG on 2-OG-dependent dioxygenases were barely observed in the testis, the LDHA protein level was remarkably decreased in Ldhc-deficient sperm, indicating that LDHC is required for LDHA expression in the sperm. This unique functional interaction between LDH family members supports lactate dehydrogenase activity in the sperm. The severely impaired motility of Ldhc-deficient sperm suggests a substantial contribution of glycolysis to energy production for sperm motility.

Patients Seventy-one and 73 years-old males visited a perioperative oral care support center to receive perioperative oral management during tongue cancer (T1N0M0) treatment. To improve their quality of life (QOL) during brachytherapy while preventing radiation-related complications including osteoradionecrosis due to 198Au grain brachytherapy, spacers for their maxilla and mandible were designed with consideration of wearing condition at an isolation ward. The spacer was created with unilateral design and with consideration of the tongue mobility during day and night. Then, the spacer was thickened on the plaster model, demonstrating the cancer lesion in the tongue in order to secure the distances from the mandibular body, maxilla and sublingual gland to the radiation sources embedded in the tongue. Discussion Tongue impression made the spacers as small as possible by thickening just around the cancer lesions so that the patients could wear them comfortably, while keeping adequate distance between the radiation sources and peripheral normal tissues. Breakable hard materials were avoided so that the patients were able to utilize the spacers safely without accidentally swallowing a broken fragment. Additionally, considering the upward movement of the tongue in a sleeping posture, the upper spacers were also prepared to protect the maxillae. Computer simulation revealed that the design of our spacers had enough effect on a reduction in radiation to prevent osteoradionecrosis in the maxilla as well as mandibular body. Conclusions This report demonstrated the importance of the spacers created with consideration of patients’ wearing condition to improve their QOL during brachytherapy.

Acetaldehyde is a potential carcinogen for esophageal cancer, and some oral microorganisms produce acetaldehyde from ethanol or glucose. In this prospective study, we examined the influence of professional oral care on acetaldehyde levels in mouth air of esophageal cancer patients. Acetaldehyde concentrations in mouth air and breath were measured by a portable gas chromatograph, and acetaldehyde production from oral microbiota was also evaluated. Samples were taken from 21 esophageal cancer patients (median age 68 years) and 20 age-matched healthy volunteers (control group) before and after oral care. Post-operative samples were also taken from 17 patients who had undergone surgery. All samples (mouth air, breath, and saliva) were collected 2 to 3 hours after lunch. Oral microbial samples were prepared from saliva. Genotype analysis of alcohol dehydrogenase 1B (ADH1B) and aldehyde dehydrogenase-2 (ALDH2) genes revealed no significant differences in the genotypes between the two groups. In the control group, acetaldehyde levels in mouth air showed no significant changes after oral care, while the amount of microbial acetaldehyde production from ethanol was significantly decreased. By contrast, among the patients, acetaldehyde levels in mouth air were significantly decreased after oral care and after operation, while the amount of microbial acetaldehyde production from ethanol showed no significant changes. Moreover, microbial acetaldehyde production from glucose was significantly decreased after operation. Overall, oral health was poorer in the patient group. In conclusion, professional oral care for esophageal cancer patients is effective for reducing acetaldehyde levels in mouth air due to the reduction of microbial count.

Background Cleft lip and/or palate (CL/P) is among the most prevalent congenital birth defects. They negatively affect maternal psychological status and may consequently result in higher prevalence of child maltreatment. However, the association of CL/P births with bonding disorders still remains unclear. To address this question, we examined the impact of CL/P birth on mother-to-infant bonding, using the nationwide birth cohort study, Japan Environment and Children's Study. Methods This study was conducted as a nationwide birth cohort study of the Japan environment and children’s study (JECS), an ongoing nationwide birth cohort study in Japan. 104,065 of foetuses in fifteen regional centres in JECS were enrolled. Finally, the participants consisted of 79,140 mother-infant pairs, of which 211 mothers with CL/P infants were included in our analyses. Results First, no increased risk of bonding disorders was observed among all the mothers with CL/P births (odds ratio [95% CI]; 0.97 [0.63-1.48], p = 0.880), and advanced maternal age or multiple parity would adversely affect the associations between bonding disorders and CL/P births, respectively. Thus, after stratification with a combination of maternal age and parity, a significant association of CL/P birth with bonding disorders was found only among advanced-age multiparae (OR [95% CI] = 2.51 [1.17-5.37], p = 0.018), but it was weakened after additional adjustment for maternal depression. Conclusion CL/P birth may increase the risk of bonding disorders among advanced-age multiparae possibly through maternal depression. This finding provides valuable information for the provision of multidisciplinary cleft care.

One of the most severe complications of lung resection is postoperative pneumonia, and its prevention and prediction are critical. Exhaled acetone and isoprene are thought to be related to metabolism; however, little is known on their relationship with bacteria living in the oral cavity or their meaning in the acute phase in perioperative lung cancer patients. We measured acetone and isoprene in exhaled breath of 13 Japanese patients with lung cancer (3 women and 10 men, age range 62-82 years, mean 72.4 years) before breakfast during hospitalization, and compared with two acute-phase proteins, C-reactive protein (CRP) and albumin in blood serum, as well as the total number of bacteria in saliva and their activity to produce acetone and isoprene. Before operation, intensive oral care was carried out for each patient to prevent postoperative pneumonia, and swallowing and cough reflexes were measured for 12 of 13 patients to assess risk of postoperative pneumonia. Breath and saliva were sampled before intensive oral care (T1), after oral care but before operation (T2), and after operation (T3) during hospitalization. The total number of oral bacteria in saliva decreased significantly from T1 to T2 among 13 patients. No acetone or isoprene was detected from saliva after in vitro incubation under anaerobic or aerobic conditions, but both acetone and isoprene were detected in breath. After operation, breath acetone correlated significantly with CRP (Spearman's ρ = 0.559, P = 0.03), but not with albumin. Breath isoprene correlated significantly with albumin (Spearman's ρ = 0.659, P = 0.008), but not with CRP after operation. Although the number of subjects was small, our results support the hypothesis that breath acetone and isoprene may be related with these acute-phase proteins, which reflect inflammatory reactions and subsequent changes in metabolism in the early postoperative phase of lung resection.

Additional file 3: Table S3. Association of bonding disorders with CL/P among advanced-age multiparae in complete dataset analysis

Background Since most oral malodor originates from microbial activities in the mouth, the role of microorganisms in producing malodorous gases has been clarified by numerous studies, accompanied by the development of analytical techniques for treatment of halitosis. Highlight Oral microorganisms should be controlled to prevent aspiration pneumonia, especially for elderly perioperative patients. Malodorous gases from the mouth can be an indicator of oral or systemic conditions among patients in intensive care units. Recently, malodorous gases originating from oral microorganisms have been reported as a causal factor in carcinogenesis. Conclusion Further analysis of oral malodor might be useful in accessing the risk of aspiration pneumonia and oral cancer.

Seventy-one and 73 years-old males visited a perioperative oral care support center to receive perioperative oral management during tongue cancer (TTongue impression made the spacers as small as possible by thickening just around the cancer lesions so that the patients could wear them comfortably, while keeping adequate distance between the radiation sources and peripheral normal tissues. Breakable hard materials were avoided so that the patients were able to utilize the spacers safely without accidentally swallowing a broken fragment. Additionally, considering the upward movement of the tongue in a sleeping posture, the upper spacers were also prepared to protect the maxillae. Computer simulation revealed that the design of our spacers had enough effect on a reduction in radiation to prevent osteoradionecrosis in the maxilla as well as mandibular body.This report demonstrated the importance of the spacers created with consideration of patients' wearing condition to improve their QOL during brachytherapy.

The transcription factor Bach2 regulates both acquired and innate immunity at multiple steps, including antibody class switching and regulatory T cell development in activated B and T cells, respectively. However, little is known about the molecular mechanisms of Bach2 regulation in response to signaling of cytokines and antigen. We show here that mammalian target of rapamycin (mTOR) controls Bach2 along B cell differentiation with two distinct mechanisms in pre-B cells. First, mTOR complex 1 (mTORC1) inhibited accumulation of Bach2 protein in nuclei and reduced its stability. Second, mTOR complex 2 (mTORC2) inhibited FoxO1 to reduce Bach2 mRNA expression. Using expression profiling and chromatin immunoprecipitation assay, the Ccnd3 gene, encoding cyclin D3, was identified as a new direct target of Bach2. A proper cell cycle was lost at pre-B and mature B cell stages in Bach2-deficient mice. Furthermore, AZD8055, an mTOR inhibitor, increased class switch recombination in wild-type mature B cells but not in Bach2-deficient cells. These results suggest that the mTOR-Bach2 cascade regulates proper cell cycle arrest in B cells as well as immunoglobulin gene rearrangement.

Background Most bone metastases are observed in the trunk of the body. Metastasis in the mandibular condyle is rare. In many case reports, temporary common temporomandibular joint disorder-like symptoms can be a sign of relapse and metastasis. Case presentation We report a rare case of breast carcinoma metastatic to the left mandibular condyle in a 55-year-old Japanese woman, who visited our department for a dental check-up prior to chemotherapy. She had almost no symptoms, but radiographs suggested the existence of metastasis. Conclusions In many case reports, patients had some symptoms. In this case report, our patient had slight symptoms, but we were able to confirm the metastasis from the symptoms and panoramic dental radiograph. When patients complain about discomfort of the temporomandibular joint, we need to consider the possibility of metastasis and notice changes on the panoramic dental radiograph.

On March 11, 2011, Japan experienced an earthquake of magnitude 9.0 and subsequent enormous tsunamis. This disaster destroyed many coastal cities and caused nearly 20,000 casualties. In the aftermath of the disaster, many tsunami survivors who lost their homes were forced to live in small temporary apartments. Although all tsunami survivors were at risk of deteriorating health, the elderly people were particularly at a great risk with regard to not only their physical health but also their mental health. In the present study, we performed a longitudinal cohort study to investigate and analyze health conditions and cognitive functions at 28, 32, and 42 months after the disaster in the elderly people who were forced to reside in temporary apartments in Kesennuma, a city severely damaged by the tsunamis. The ratio of people considered to be cognitively impaired significantly increased during the research period. On the other hand, the mean scores of the Kessler Psychological Distress Scale-6 and Athens Insomnia Scale improved based on the comparison between the data at 24 and 42 months. The multiple logistic regression analysis revealed that frequency of “out-of-home activities” and “walking duration” were independently associated with an increase in the ratio of people with cognitive impairment. We concluded that the elderly people living in temporary apartments were at a high risk of cognitive impairment and “out-of-home activities” and “walking” could possibly maintain the stability of cognitive functions.

Current understanding of the pathogenic etiology of supra- and subgingival plaque assumes dynamic changes of pathogenic potential depending upon the crosstalk among resident biofilm, as well as that among microbial communities and host tissues, called the ecological plaque hypothesis. Manipulation of bacterial and environmental factors, which affect the proportion and virulence potential, has potential for the control of the pathogenicity of biofilm. Most periodontal pathogens, except Actinobacillus actinomycetemcomitans , are oxygen-sensitive, proteolytic species and are thus unable to survive inside the plaque, which is sufficiently thin to allow oxygen exposure, and also lacks a nutritional supply of protein and some other essential factors. A preventive strategy against these pathogenic microbes would be an ecological approach to maintain health-compatible microflora on the surface of teeth. On the other hand, the strategy against A. acinomycetemcomitans , which is the microbe to which Koch's postulate is most applicable, would be employed in an approach to eliminate this pathogen from the microbial community by use of antibiotics or the humoral immune response of the host. For accomplishing both strategies, we discuss the host–parasite interface, not only the interaction but also the ecological and structural factors, for the purpose of preventing the onset and progression of periodontal diseases.

OBJECTIVES CD14, toll-like receptor 4 (TLR4) and MyD88 have been shown to mediate responsiveness in host cells to lipopolysaccharide. We investigated here the regulatory effects of inflammatory cytokines on the expression of membrane CD14 (mCD14), TLR4 and MyD88, and on subsequent responsiveness to lipopolysaccharide from Actinobacillus actinomycetemcomitans in human gingival fibroblasts. MATERIALS AND METHODS Following treatment with either interleukin-1beta, tumor necrosis factor-alpha (TNF-alpha) or gamma-interferon (IFN-gamma), expression of mCD14/TLR4 and MyD88 was determined by flow cytometry and western blotting, respectively. After pretreatment with IFN-gamma, cells were pre-incubated with either anti-CD14 antibody MY4 or anti-TLR4 antibody HTA125 and subsequently treated with A. actinomycetemcomitans lipopolysaccharide. Then, phosphorylation of mitogen-activated protein (MAP) kinases and IkappaBalpha was examined by western blotting, and production of interleukin-6 and interleukin-8 was measured by their respective enzyme-linked immunosorbent assay (ELISA) kits. RESULTS IFN-gamma stimulated expression of mCD14, whereas -1beta and TNF-alpha did not. Expression of MyD88 but not TLR4 was also enhanced by IFN-gamma. The lipopolysaccharide activated MAP kinases, such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and p38, and IkappaBalpha and stimulated production of interleukin-6 and interleukin-8. The lipopolysaccharide-stimulated interleukin-6 and interleukin-8 production was markedly inhibited by MY4 or HTA125. Pretreatment with IFN-gamma augmented the following activation of MAP kinases and IkappaBalpha and production of interleukin-6 and interleukin-8 in response to the lipopolysaccharide. CONCLUSIONS These results suggest that the augmentation by IFN-gamma of the responsiveness to A. actinomycetemcomitans lipopolysaccharide, such as activation of MAP kinases and IkappaBalpha and terminal cytokine production in human gingival fibroblasts, may be partially mediated by up-regulation of CD14 and MyD88 expression.

Human papillomavirus type 16 (HPV16), a causative agent of cervical cancers, encodes the E6 and E7 oncogenes, whose simultaneous expression is pivotal for malignant transformation and maintenance of malignant phenotypes. In the hope of developing a gene-specific therapy for HPV-related cancer, we examined the effects of E6 short-interfering RNA (siRNA) on the expression of these oncogenes and on the cell growth of HPV16-related cervical cancer cells. Using SiHa cervical cancer cells, we demonstrated that E6 siRNA decreased the levels of mRNA encoding E6 as well as that encoding E7 protein and also induced nuclear accumulation of p53, the most important target of E6. E6 siRNA suppressed monolayer and anchorage-independent growth of SiHa cells, which was associated with p21(CIP1/WAF1) induction and hypophosphorylation of retinoblastoma protein. Further, SiHa cells treated with E6 siRNA formed tumors in NOD/SCID mice that were significantly smaller than in those treated with control siRNA. Our results show HPV E6 siRNA as a candidate for gene-specific therapy for HPV-related cervical cancer.

Objectives and background: Endothelin-1 (ET-1) is a 21-amino acid peptide with multifunctional regulation. ET-1 expresses in various cells during inflammation. The present study aimed to examine the ET-1 expression in oral epithelial cells after infection with the periodontal pathogen and to investigate the presence of ET-1 in human inflamed and uninflamed gingival tissues. Materials and methods: The KB cells were infected with Porphyromonas gingivalis and the expression level of ET-1 was examined using reverse transcription-polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA). The immunohistochemical analysis of ET-1 was performed in gingival tissues obtained from patients. In addition, the ET-1 mRNA expression in each tissue was also investigated by RT-PCR. Results: The expression of ET-1 in KB cells was strongly induced by the P. gingivalis infection. On the other hand, the strong immunoreactivity for ET-1 was observed in the epithelium and vascular endothelial cells of the inflamed gingival tissue. Furthermore, the level of ET-1 mRNA was greater in the inflamed tissues. Conclusion: These results suggested that the expression level of ET-1 in gingival epithelial cells might be enhanced during the periodontal inflammation.

Bone morphogenetic proteins, including growth/differentiation factor-5 (GDF-5), are multifunctional cytokines. Recent studies of intracellular signal transduction mechanisms for the transforming growth factor-beta superfamily have focused on Smad proteins. However, scant attention has been given to the mechanism by which GDF-5 exerts its negative growth effect on immunological competent cells. In the present study, we demonstrated that GDF-5 induced cell cycle arrest in the G1 phase before the appearance of apoptosis in mouse B cell hybridoma HS-72 cells, while the ectopic expression of Smad6 and Smad7 in HS-72 cells suppressed the GDF-5-induced G1 cell cycle arrest by abolishing the expression of p21(CIP-1/WAF-1) and hypophosphorylation of retinoblastoma protein. Moreover, we found that Smad6 and Smad7 suppressed GDF-5-induced apoptosis in HS-72 cells. These findings indicated that Smad6 and Smad7 exhibit inhibitory effects toward GDF-5-mediated signaling in B lineage cells.

Recent studies have revealed that both transforming growth factor-beta (TGF-beta) and activin A play pivotal roles in osteoclastogenesis. In this report, we show that the effect of TGF-beta family members, TGF-beta1 and activin A, but not BMP-2, enhance multinucleated osteoclast-like cell (OCL) formation induced by receptor activator of NF-kappaB ligand (RANKL) in isolated bone marrow macrophages and monocytic cell line, RAW264.7. TGF-beta1 and activin A caused the growth suppression and concomitant expression of tartrate-resistant acid phosphatase (TRAP) and c-Src, without inducing syncytium formation or increasing the survival rate in RAW264.7 cells. Although TGF-beta1 and activin A had no effect on NF-kappaB and JNK activities, these factors enhanced the expression of JunB, a component of the AP-1 transcriptional complex. These results suggest that TGF-beta1 and activin A may function as commitment factors in osteoclastic differentiation, not as a crucial component for terminal differentiation to form multinucleated OCLs nor in OCL survival.

BMPs exert a negative growth effect on various types of cells. We have previously reported that BMP-2 inhibited the growth of HS-72 mouse hybridoma cells by inducing p21(CIP1/WAF1) expression. In the present study, we demonstrated that BMP-2 activated the mouse p21(CIP1/WAF1) promoter in HS-72 cells, and that a 29-base pair (b) region of the promoter (-1928/-1900 relative to the TATA box), conserved between mice and humans, was responsive to BMP-2 as well as expression of Smad1, Smad4, and constitutively active mutants of BMP type I receptors. Furthermore, an oligonucleotide containing the 29-b region was found to be associated with Smad4 and phosphorylated Smad1 in the nuclear extract of BMP-2-stimulated HS-72 cells. These results suggested that BMP-2 might activate p21(CIP1/WAF1) transcription by inducing a binding of Smad4 and Smad1 to the 29-b region in HS-72 cells.

Infection of murine macrophages in vitro with periodontopathic bacterium Actinobacillus actinomycetemcomitans induces apoptotic cell death. In this study, we investigated the involvement of caspases in apoptotic cell death of A. actinomycetemcomitans-infected macrophages. Two peptide inhibitors of caspases, benzyloxycarbonyl-Val-Ala-Asp (OMe)-fluoromethyl ketone (Z-VAD-FMK) and benzyloxycarbonyl-Asp-Glu-Val-Asp (OMe)-fluoromethyl ketone (Z-DEVD-FMK), inhibited apoptotic cell death of murine macrophage cell line J774.1 infected with A. actinomycetemcomitans. During the process of apoptosis, interleukin-1beta (IL-1beta) was detected in the culture supernatants of J774.1 cells. IL-1beta secretion was blocked by the caspase-1 inhibitor, Z-VAD-FMK, indicating that caspase-1 is involved in not only the induction of apoptosis but also the IL-1beta secretion from A. actinomycetemcomitans-infected J774.1 cells. Immunoblot analysis revealed that the infection of A. actinomycetemcomitans to J774.1 cells induced the cleavage of retinoblastoma protein (Rb), suggesting that caspase-3 was activated by A. actinomycetemcomitans infection. The cytosol from A. actinomycetemcomitans-infected J774.1 cells induced Rb proteolysis in vitro, which was inhibited by the caspase-3 inhibitor, Z-DEVD-FMK. Furthermore, caspase-3-like activity was markedly increased in J774.1 cells infected with A.actinomycetemcomitans between 12 h and 24 h, which was subsequently inhibited by the addition of caspase-3 inhibitor, Z-DEVD-FMK. These findings indicate that caspase-3 induces apoptosis in J774.1 cells infected with A. actinomycetemcomitans. Taken together, these results suggest that caspase-1 and caspase-3 are involved in the induction of apoptosis in A. actinomycetemcomitans-infected macrophages.

Osteoclasts, bone-resorbing multinucleated cells, develop from monocyte-macrophage lineage cells in the presence of osteoclast differentiation factor (ODF, also called RANKL/TRANCE/OPGL) and macrophage colony-stimulating factor (M-CSF). M-CSF-dependent bone marrow macrophages (M-BMMPhis) from mouse bone marrow cells have been shown to differentiate into osteoclast-like multinucleated cells (OCLs) in the presence of soluble ODF/RANKL (sODF/RANKL) and M-CSF within 3 days. In this study, we found that stimulation of M-BMMPhis with sODF/RANKL induced a transient expression of cyclin-dependent kinase inhibitors (CDK inhibitors) p21(WAF1/CIP1) and p27(KIP1) by 24 h. The CDK inhibitor proteins disappeared by 48 h. Tumor necrosis factor alpha (TNF-alpha), which is reported to stimulate OCL differentiation, stimulated p21(WAF1/CIP1) and p27(KIP1) expression in M-BMMPhis as well. However, M-CSF alone did not stimulate the expression of the two CDK inhibitors. To clarify the role of p21(WAF1/CIP1) and p27(KIP1) in osteoclastogenesis, accumulation of these CDK inhibitors was aborted by antisense oligonucleotides. Treatment with p21(WAF1/CIP1) antisense oligonucleotide alone, or p27(KIP1) antisense oligonucleotide alone, showed a limited inhibitory effect on OCL formation. However, treatment with a mixture of these two antisense oligonucleotides strongly inhibited OCL formation. These results suggest that a combined modulation of the CDK inhibitors p21(WAF1/CIP1) and p27(KIP1) may be involved in osteoclast differentiation induced by ODF/RANKL.

Bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)–β superfamily, are a group of related proteins that are capable of inducing the formation of cartilage and bone but are now regarded as multifunctional cytokines. We show in this report a novel function of BMPs in hematopoietic cells: BMP-2 induces apoptosis not only in human myeloma cell lines (U266, RPMI 8226, HS-Sultan, IM-9, OPM-2, and KMS-12 cells), but also in primary samples from patients with multiple myeloma. The mechanism of BMP-2–induced apoptosis was investigated with the use of U266 cells, which are dependent on the interleukin-6 autocrine loop. We showed that BMP-2 caused cell-cycle arrest in the G1 phase and the subsequent apoptosis of myeloma cells. BMP-2 up-regulated the expression of cyclin-dependent kinase inhibitors (p21CIP1/WAF1 and p27KIP1) and caused hypophosphorylation of retinoblastoma (Rb) protein. In studies of apoptosis-associated proteins, BMP-2 was seen to down-regulate the expression of Bcl-xL; however, BMP-2 had no effects on the expression of Bcl-2, Bax, or Bad. Therefore, BMP-2 induces apoptosis in various human myeloma cells by means of the down-regulation of Bcl-xL and by cell-cycle arrest through the up-regulation of p21CIP1/WAF1 and p27KIP1 and by the hypophosphorylation of Rb. Further analysis showed that the signal transducer and activator of transcription 3 (STAT3) was inactivated immediately after BMP-2 treatment. We conclude that BMP-2 would be useful as a novel therapeutic agent in the treatment of multiple myeloma both by means of its antitumor effect of inducing apoptotis and through its original bone-inducing activity, because bone lesions are frequently seen in myeloma patients.

Nod1 is an Apaf-1-like molecule composed of a caspase-recruitment domain (CARD), nucleotide-binding domain, and leucine-rich repeats that associates with the CARD-containing kinase RICK and activates nuclear factor kappaB (NF-kappaB). We show that self-association of Nod1 mediates proximity of RICK and the interaction of RICK with the gamma subunit of the IkappaB kinase (IKKgamma). Similarly, the RICK-related kinase RIP associated via its intermediate region with IKKgamma. A mutant form of IKKgamma deficient in binding to IKKalpha and IKKbeta inhibited NF-kappaB activation induced by RICK or RIP. Enforced oligomerization of RICK or RIP as well as of IKKgamma, IKKalpha, or IKKbeta was sufficient for induction of NF-kappaB activation. Thus, the proximity of RICK, RIP, and IKK complexes may play an important role for NF-kappaB activation during Nod1 oligomerization or trimerization of the tumor necrosis factor alpha receptor.

We have previously found that bone morphogenetic protein-2 (BMP-2), a member of the transforming growth factor-β family, induces cell-cycle arrest in the G1 phase and apoptotic cell death of HS-72 mouse hybridoma cells. In this study, we show that BMP-2 did not alter expression of cyclin D, cyclin E, cyclin-dependent kinase 2 (CDK2), CDK4, p27 KIP1 , p16 INK4a , or p15 INK4b , but enhanced expression of p21 CIP1/WAF1 . Accumulation of p21 CIP1/WAF1 resulted in increased binding of p21 CIP1/WAF1 to CDK4 and concomitantly caused a profound decrease in the in vitro retinoblastoma protein (Rb) kinase activity of CDK4. Furthermore, the ectopic expression of human papilloma virus type-16 E7, an inhibitor of p21 CIP1/WAF1 and Rb, reverted G1 arrest induced by BMP-2. Expression of E6/E7, without increasing the p53 level, blocked inhibition of Rb phosphorylation and G1 arrest, but did not attenuate cell death in BMP-treated HS-72 cells. Taken together, these results suggest that inhibition of Rb phosphorylation by p21 CIP1/WAF1 is responsible for BMP-2-mediated G1 arrest and that BMP-2-induction of apoptosis might be independent of Rb hypophosphorylation.

The DNA fragmentation factor (DFF) is composed of two subunits, the 40-kDa caspase-3-activated nuclease (DFF40/CAD) and its 45-kDa inhibitor (DFF45/ICAD). During apoptosis, DFF-40/CAD is activated by caspase-3-mediated cleavage of DFF45/ICAD. Mutational analysis of DFF40/CAD revealed that DFF40/CAD is composed of a C-terminal catalytic domain and an N-terminal regulatory domain. Deletion of the catalytic domain (residues 290-345) abrogated the caspase-3-induced nuclease activity of DFF40/CAD but not its ability to interact with DFF45/ICAD. Conversely, removal of the regulatory domain (residues 1-83) yielded a constitutively active DFF40/CAD nuclease that neither bound to its inhibitor nor required caspase-3 for activation. Amino acid alignment revealed that the regulatory domain of DFF40/CAD has homology to the N-terminal region of mammalian and Drosophila DFF45/ICAD and CIDE-N, a regulatory domain previously identified in pro-apoptotic CIDE proteins. Mutational analysis of the N-terminal region revealed mutants with diminished nuclease activity but with intact ability to bind DFF45/ICAD. Thus, CIDE-N represents a new type of domain that is associated with the regulation of the apoptosis/DNA fragmentation pathway.

We have identified and characterized Diva, which is a novel regulator of apoptosis. Sequence analysis revealed that Diva is a member of the Bcl-2 family of proteins containing Bcl-2 homology domain 1, 2, 3, and 4 (BH1, BH2, BH3, and BH4) regions and a carboxyl-terminal hydrophobic domain. The expression of Diva mRNA was detected in multiple embryonic tissues but was restricted to the ovary and testis in adult mice. The expression of Diva promoted the death of 293T, Ramsey, and T47D cells as well as that of primary sensory neurons, indicating that Diva is a proapoptotic protein. Significantly, Diva lacks critical residues in the conserved BH3 region that mediate the interaction between BH3-containing proapoptotic Bcl-2 homologues and their prosurvival binding partners. Consistent with this, Diva did not bind to cellular Bcl-2 family members including Bcl-2, Bcl-XL, Bcl-w, Mcl-1, and A1/Bfl-1. Furthermore, mutants of Diva lacking the BH3 region fully retained their proapoptotic activity, confirming that Diva promotes apoptosis in a BH3-independent manner. Significantly, Diva interacted with a viral Bcl-2 homologue (vBcl-2) encoded by the Kaposi's sarcoma-associated herpesvirus. Consistent with these associations, apoptosis induced by Diva was inhibited by vBcl-2 but not by Bcl-XL. Importantly, Diva interacted with Apaf-1, an adapter molecule that activates caspase-9, a central death protease of the apoptotic pathway. The expression of Diva inhibited the binding of Bcl-XL to Apaf-1, as determined by immunoprecipitation assays. Thus, Diva represents a novel type of proapoptotic Bcl-2 homologue that promotes apoptosis independently of the BH3 region through direct binding to Apaf-1, thus preventing Bcl-XL from binding to the caspase-9 regulator Apaf-1.

Activins transduce their signals by binding to activin type I receptors and activin type II receptors, both of which contain a serine/threonine kinase domain. In this study, we established stable transfectants expressing two types of activin receptors, ActRI and ActRIB, to clarify the role of these receptors in activin signalling for growth inhibition in HS-72 mouse B-cell hybridoma cells. Over-expression of ActRI suppressed activin A-induced cell-cycle arrest in the G1 phase caused by inhibition of retinoblastoma protein phosphorylation through induction of p21CIP1/WAF1, a cyclin-dependent kinase inhibitor, and subsequent apoptosis. In contrast, HS-72 clones that over-expressed ActRIB significantly facilitated activin A-induced apoptosis. These results indicate that ActRI and ActRIB are distinct from each other and that the ActRI/ActRIB expression ratio could regulate cell-cycle arrest in the G1 phase and subsequent apoptosis in HS-72 cells induced by activin A.

Actinobacillus actinomycetemcomitans is considered to be an aetiological agent in various forms of periodontitis, with serotype b-specific carbohydrate being the immunodominant antigen of A. actinomycetemcomitans Y4 in high-responder patients. Lipopolysaccharide (LPS) of the organism may also be an important antigen. The purpose of the present study was to clarify the importance of LPS as an antigen of A. actinomycetemcomitans. Twenty patients who had high antibody titres to strain Y4 were selected, and the reactivity of their sera with LPS was determined by ELISA and Western blotting. Two groups of patients were observed: group 1 had high IgG titres only to serotype b strain, whereas group 2 had high IgG titres to serotypes a, b and c strains. The results of adsorption tests showed that anti-A. actinomycetemcomitans Y4 antibody in group 1 patients mostly consisted of antibody reactive with the serotype b-specific carbohydrate, whereas the antibody in group 2 patients mostly consisted of antibody reactive with the LPS of all serotypes. These data show that anti-LPS antibody is present and predominant in anti-A. actinomycetemcomitans Y4 antibody from some high-responder patients, and indicate an important role for LPS as an antigen in the humoral immune response to the organism.

DFF45 is a subunit of the DNA fragmentation factor (DFF) that is cleaved by caspase-3 during apoptosis. However, the mechanism by which DFF45 regulates apoptotic cell death remains poorly understood. Here we report the identification and characterization of two mammalian genes, CIDE-A and CIDE-B, encoding highly related proteins with homology to the N-terminal region of DFF45. CIDE-A and CIDE-B were found to activate apoptosis in mammalian cells, which was inhibited by DFF45 but not by caspase inhibitors. Expression of CIDE-A induced DNA fragmentation in 293T cells, which was inhibited by DFF45, further suggesting that DFF45 inhibits the apoptotic activities of CIDEs. In addition to mammalian CIDE-A and CIDE-B, we identified DREP-1, a Drosophila melanogaster homolog of DFF45 that could inhibit CIDE-A-mediated apoptosis. Mutant analysis revealed that the C-terminal region of CIDE-A was necessary and sufficient for killing whereas the region with homology to DFF45 located in the N-terminus was required for DFF45 to inhibit CIDE-A-induced apoptosis. CD95/Fas-mediated apoptosis was enhanced by CIDEs but inhibited by DFF45. These studies suggest that DFF45 is evolutionarily conserved and implicate CIDEs as DFF45-inhibitable effectors that promote cell death and DNA fragmentation.

We previously identified an IS200-like sequence (ISAa1) in the genome of Actinobacillus actinomycetemcomitans FDC Y4. One or more hybridizing bands to the ISAa1 probe were detected in each of several reference strains, representing three of the serotypes (a through c) of A. actinomycetemcomitans. In this study, we examined whether a restriction fragment-length polymorphism (RFLP) with ISAa1 as a probe could differentiate clinical isolates. One or more hybridizing bands were detected in each of the 27 strains examined, which could be divided into seven groups according to restriction fragment-length polymorphism pattern. Several strains were observed with identical restriction fragment-length polymorphism types but with different serotypes. Conversely, strains were also observed with differing restriction fragment-length polymorphism types and identical serotypes.

B-1 cells are physically and functionally unique B cells that produce polyreactive natural antibody. This study examined the activation of B-1 cells in inflamed gingival tissue. Serum IgG antibodies to phosphorylcholine, E. coli LPS, DNA, and some commensal bacteria were examined in adult periodontitis patients and healthy subjects. In addition, the proportion of B-1a (CD20+CD5+) cells and the amount of IL-6 and IL-10 in the inflamed gingival tissues were examined. The serum levels of IgG antibodies to phosphorylcholine, E. coli LPS, and commensal bacteria were significantly higher in the adult periodontitis patients than the healthy subjects. The proportion of B-1a cells and the amount of IL-6 and IL-10 were significantly higher in the inflamed gingival tissues than in peripheral blood from the healthy subjects. These results suggest the activation of B-1 cells in the inflamed gingival tissue of adult periodontitis patients, and that B-1 cells may serve as the first line of defense by producing polyreactive antibodies to phosphorylcholine, LPS, and commensal bacteria.

We have previously demonstrated that cell death of WEHI-231 cells induced by specific inhibitors of vacuolar type H+-ATPase (V-ATPase) occurs through apoptosis. CD40 is involved in regulating activation, differentiation, and apoptosis of B cells. Here we show that the CD40 ligation rescues WEHI-231 cells from apoptotic cell death induced by a specific V-ATPase inhibitor, concanamycin A. CD40 signaling with anti-CD40 antibody resulted in the induction of Bcl-2 and Bcl-XL proteins in WEHI-231 cells. Constitutive expression of Bcl-2 but not Bcl-XL inhibited concanamycin A-induced apoptosis. These findings suggest that the expression of Bcl-2 mediated through CD40 signaling rescues the apoptotic cell death induced by blockade of V-ATPase. Interestingly, the acidification of intracellular acidic compartments was completely inhibited when WEHI-231 cells were cultured with concanamycin A, even in the presence of anti-CD40 antibody. In addition, apoptosis in WEHI-231 cells induced by concanamycin A was strongly suppressed when cultured with imidazole, a cell-permeable base, suggesting that apoptosis induced by concanamycin A is preceded by intraacidification.

Activins, members of the transforming growth factor-beta family, have been implicated in the regulation of growth and differentiation of various types of cells. We have recently found that activin A induces apoptotic cell death of plasmacytic cells including B cell hybridoma cells and myeloma cells. In the present study, we demonstrated that activin A caused cell-cycle arrest in the G1 phase before appearance of apoptotic cells in mouse B cell hybridoma cells. Phosphorylation of retinoblastoma protein (Rb) and in vitro Rb kinase activity of cyclin-dependent kinase (CDK)4 was inhibited in activin A-treated cells. Analysis of expression of genes regulating Rb phosphorylation revealed that activin A suppressed cyclin D2, the sole D-type cyclin gene expressed in the hybridoma cells, and activated p21CIP1/WAF1 but had no effect on expression of cyclin-dependent kinases (CDK2, CDK4, CDK6) and other CDK inhibitors (p27KIP1, p16INK4a, p15INK4b). Modulation of cyclin D2 and p21CIP1/WAF1 expression resulted in a decrease in level of cyclin D2-CDK4 complex and an increase in level of CDK4 complexed with p21CIP1/WAF1. Moreover, overexpression of cyclin D2 partially abrogated inhibition of Rb phosphorylation and G1 arrest in the hybridoma cells.

We report the evidence for apoptosis in J774.1 cells by the periodontopathic bacterium Actinobacillus actinomycetemcomitans, suggesting that the ability of A. actinomycetemcomitans to promote apoptosis might be important in the initiation and development of periodontitis. In this study, we examined the role of macrophage CD14, anchored by a glycerophosphatidylinositol tail, in the induction of apoptosis by A. actinomycetemcomitans infection by using the parent J774.1 cells and CD14-defective mutant (LR-9) cells. A small number of A. actinomycetemcomitans Y4 cells inside the LR-9 cells compared with the number in J774.1 cells was detected by confocal scanning microscopy. We found that LR-9 cells showed a weak cytotoxic effect after being infected with A. actinomycetemcomitans Y4. Apoptotic cell death of LR-9 cells infected with A. actinomycetemcomitans Y4, compared with that of the parent J774.1 cells was almost undetectable, as shown by the proportion of fragmented DNA in agarose gel electrophoresis and by the terminal deoxynucleotidyl transferase-mediated dUTP end-labeling method. Flow cytometric cell cycle analysis of J774.1 cells infected with A. actinomycetemcomitans Y4 revealed the increased percentage of apoptotic cells with hypodiploid DNA. However, LR-9 cells infected with A. actinomycetemcomitans Y4 showed no increase in population of apoptotic nuclei compared with the noninfected cells. These findings suggest that the CD14 molecules may contribute to the phagocytosis of A. actinomycetemcomitans by J774.1 cells and regulate, at least in part, apoptotic cell death of macrophages infected with A. actinomycetemcomitans.

In order to cultivate fermentative treponemes isolated on Medium 10 with the plate-in-bottle method from subgingival plaque, we developed the enriched Medium 10. Enriched Medium 10 broth consisted mainly of Trypticase peptone (2 g/1), yeast extract (10 g/1), glucose (5 g/1) and volatile fatty acids mixture (3.1 ml/1). The growth curves in enriched Medium 10 broth showed that clinical isolates of Treponema socranskii reached the early stationary phase on the 3rd–5th day, and the doubling times averaged 22.2 h. By using enriched Medium 10, the nutritional analysis of oral treponemes was examined for each compound of the volatile fatty acid and various sera. Clinical isolates were found to require iso-butyric acid, 2-methylbutyric acids and/or iso-caproic acid independently for their growth. However, the growth of these fermentative treponemes was inhibited by addition of various sera to this medium.

Objectives To identify factors associated with dry mouth. Background Dry mouth adversely affects oropharyngeal health, particularly in elderly, and can lead to pneumonia. A better understanding of the epidemiology of dry mouth is therefore important in improving treatment strategies and oral health in high-risk elderly patients. Methods We conducted a cross-sectional study involving 383 dependent Japanese elderly individuals (65–84 [n = 167] and ≥85 [n = 216] years) at eight long-term care facilities and hospitals. Thirty-four potential factors associated with dry mouth were examined by multiple logistic regression analysis. The primary outcome was dry mouth, as diagnosed by tongue dorsum moisture. Results We identified that body mass index and severity of physical disability were identified as a potential factors associated with dry mouth in the super-elderly (≥85 years) group, whereas severity of physical disability, outcome measurement time, high daily water consumption, mouth breathing, use of antidepressants and diuretics, and high frequency of daily brushing (≥2 times per day; Odds ratio: 5.56; 95% Confidence Interval: 1.52–20.00) were associated with dry mouth in the 65- to 84-year-old group. Conclusion To our knowledge, this is the first study to identify a link between frequency of daily brushing and dry mouth and suggests that tooth brushing should be encouraged in high-risk dependent Japanese elderly (65–84 years), particularly those taking antidepressants and/or diuretics.