Hematogenous apoptotic mechanism in salivary glands in chronic periodontitis.

OBJECTIVE: The aim of the study is to investigate the apoptotic mechanism in salivary glands in the rat experimental periodontitis model. DESIGN: A rat periodontitis model was prepared by using a ligature around the second upper molar. In the salivary (parotid and submandibular) glands and blood samples, putative apoptotic factors and pathway molecules were investigated in vivo and in vitro. RESULTS: Four weeks of ligation (chronic periodontitis) demonstrated significant apoptotic atrophy of the salivary gland, but one week of ligation (initial periodontitis) did not. In the blood plasma, tumor necrosis factor-α (TNF-α) was increased in the periodontitis model, but interleukin-1ß and -6 were not. TNF-α receptor type 1, which has an intracellular apoptotic pathway, was expressed in the salivary glands of rats. Western blot analysis of cultured rat primary salivary gland cells demonstrated that TNF-α induced cleavage of poly (ADP-ribose) polymerase (PARP) and caspase-3 in a dose-dependent manner, indicating apoptosis induction. Additionally, we found increment of circulating lymphocytes in the model. Expression of mRNA and immunoreactive cells for the B lymphocyte marker CD19 were increased in the salivary gland in the model. Western blotting showed that coculture with extracted B cells from the periodontitis model increased cleaved PARP in salivary gland cells. CONCLUSIONS: Chronic periodontitis status leads to an increase in circulating TNF-α and B lymphocyte infiltration, resulting in apoptotic atrophy of the salivary gland as a periodontitis-induced systemic response.

Evaluation of velopharyngeal function using high-speed cine-magnetic resonance imaging based on T2-weighted sequences: a preliminary study.

The objective was to introduce a new technique for visualizing the three-dimensional (3D) movements of velopharyngeal-related muscles using high-speed cine-magnetic resonance imaging (MRI) based on T2-weighted sequences. The evaluation of phonation- and water swallowing-related events was performed in 11 healthy subjects. Specifically, whether cine-MRI could precisely visualize normal velopharyngeal function during these two events was examined. The 3D movements of the soft palate, superior pharyngeal constrictor muscles, and levator veli palatini muscles were visualized in all 11 subjects. A noteworthy finding was that the magnetic resonance signals of the superior constrictor pharyngeal muscles and the levator veli palatini muscles were significantly higher during phonation and during water swallowing than at rest. This initial study suggests that the 3D movements of velopharyngeal-related muscles can be successfully and precisely visualized without side effects. The magnetic resonance signal changes seen in the superior pharyngeal constrictor and levator veli palatini muscles using the technique described here should be useful to develop better methods of evaluation of velopharyngeal function.

Green tea extract intake during lactation modified cardiac macrophage infiltration and AMP-activated protein kinase phosphorylation in weanling rats from undernourished mother during gestation and lactation.

Maternal dietary restriction is often associated with cardiovascular disease in offspring. The aim of this study was to investigate the effect of green tea extract (GTE) intake during lactation on macrophage infiltration, and activation of adenosine monophosphate (AMP)-activated protein kinase (AMPK) and serine-threonine kinase Akt (Akt) in the hearts of weanlings exposed to maternal dietary protein restriction. Pregnant Wistar rats were fed control (C) or low-protein diets (LP) throughout gestation. Following delivery, the dams received a control or a GTE-containing control diet during lactation: control diet during gestation and lactation (CC), low-protein diet during gestation and lactation (LPC), low-protein diet during gestation and 0.12% GTE-containing low-protein diet during lactation (LPL), and low-protein diet during gestation and 0.24% GTE-containing low-protein diet during lactation (LPH). The female offspring were sacrificed at day 22. Biochemical parameters in the plasma, macrophage infiltration, degree of fibrosis and expression levels of AMPK and Akt were examined. The plasma insulin level increased in LPH compared with LPC. Percentage of the fibrotic areas and the number of macrophages in LPC were higher than those in CC. Conversely, the fibrotic areas and the macrophage number in LPH were smaller (21 and 56%, respectively) than those in LPC. The levels of phosphorylated AMPK in LPL and LPH, and Akt in LPH were greater than those in LPC. In conclusion, maternal protein restriction may induce macrophage infiltration and the decrease of insulin levels. However, GTE intake during lactation may suppress macrophage infiltration and restore insulin secretion function via upregulation of AMPK and insulin signaling in weanlings.

Leptin improves fatty liver independently of insulin sensitization and appetite suppression in hepatocyte-specific Pten-deficient mice with insulin hypersensitivity.

Nonalcoholic fatty liver disease (NAFLD) is recognized as the hepatic component of the metabolic syndrome. Although NAFLD is a major cause of cirrhosis and cancer of the liver of unknown cause, no established pharmacological treatment for NAFLD has been established yet. It has been reported that leptin treatment improved fatty liver dramatically as well as insulin resistance and hyperphagia in patients with lipodystrophy. However, it is unclear whether leptin improves fatty liver independently of these metabolic improvements. We investigated the liver effect of leptin independently of insulin sensitization and appetite suppression using hepatocyte-specific Pten-deficient (AlbCrePtenff) mouse, a model of severe fatty liver with insulin hypersensitivity. Male AlbCrePtenff mice were infused subcutaneously with leptin (20 ng/g/h) for 2 weeks using osmotic minipumps. Leptin infusion effectively reduced liver weight, liver triglyceride content, and glutamate pyruvate transaminase (GPT) concentrations as well as food intake and body weight without the change of plasma insulin concentration in AlbCrePtenff mice. Pair-feeding also reduced body weight but not liver triglyceride content. Pair feeding reduced α1 and α2 AMP-activated protein kinase (AMPK) activities and PGC1α gene expression in the liver, while leptin infusion unchanged them. The present study clearly demonstrated that leptin improve fatty liver independently of insulin sensitization and suppression of food intake. It was suggested that leptin improves fatty liver by stimulation of ß-oxidation in the liver. The present study might provide a further understanding on the mechanism of metabolic effect of leptin.

Characterization of human-type monoclonal antibodies against reduced form of hemin binding protein 35 from Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE: The gram-negative anaerobe Porphyromonas gingivalis has been implicated as an important pathogen in the development of adult periodontitis, and its colonization of subgingival sites is critical in the pathogenic process. We previously identified a 35 kDa surface protein (hemin binding protein 35; HBP35) from P. gingivalis that exhibited coaggregation activity, while additional analysis suggested that this protein possessed an ability to bind heme molecules. For development of passive immunotherapy for periodontal diseases, human-type monoclonal antibodies have been prepared using HBP35 as an antigen in TransChromo mice. In the present study, we focused on a single antibody, TCmAb-h13, which is known to inhibit heme binding to recombinant HBP35. The aim of our investigation was to clarify the redox-related function of HBP35 and consider the benefits of human-type monoclonal antibodies. MATERIAL AND METHODS: To examine the antigen recognition capability of TCmAbs with immunoblotting and Biacore techniques, we used the native form as well as several Cys-to-Ser variants of recombinant HBP35. RESULTS: We found that the redox state of recombinant HBP35 was dependent on two Cys residues, (48) C and (51) C, in the thioredoxin active center (WCGxCx). Furthermore, TCmAb-h13 recognized the reduced forms of recombinant HBP35, indicating its inhibitory effect on P. gingivalis growth. CONCLUSION: Hemin binding protein 35 appears to be an important molecule involved in recognition of the redox state of environmental conditions. In addition, TCmAb-h13 had an inhibitory effect on heme binding to recombinant HBP35, thereby interfering with P. gingivalis growth.

Role of endogenous pituitary adenylate cyclase-activating polypeptide in adult hippocampal neurogenesis.

Hippocampal neurogenesis occurs throughout life in mammals and has pivotal roles in brain functions. An enriched environment stimulates hippocampal neurogenesis, but the exact mechanisms are still unclear. The present study investigated the role of pituitary adenylate cyclase-activating polypeptide (PACAP) in adult hippocampal neurogenesis under standard or enriched rearing conditions. Rearing in the enriched conditions from 4-weeks old for 4-weeks increased the survival of newly divided cells in the subgranular zone and granule cell layer of the dentate gyrus of wild-type and PACAP-knockout (PACAP-/-) mice. The increase in the survival in the granule cell layer was less in PACAP-/- mice than in the wild-type mice. In contrast, the proliferation of newly divided cells in mice reared in the standard and enriched conditions did not differ between the wild-type and PACAP-/- mice. Regarding the differentiation of newborn cells in the dentate gyrus, most of the newly divided cells exhibited the neuronal phenotype in both the wild-type and PACAP-/- mice under standard and enriched conditions. These findings suggest that endogenous PACAP is partly involved in the survival of the enriched environment-induced generation, but not in the basal rate, of newborn cells in the dentate gyrus of the adult hippocampus.

Effects of KP-496, a novel dual antagonist at the cysteinyl leukotriene receptor 1 and the thromboxane A(2) receptor, on airway obstruction in guinea pigs.

BACKGROUND AND PURPOSE: KP-496 is a novel dual antagonist for cysteinyl leukotriene receptor 1 (CysLT(1)) and thromboxane A(2) (TXA(2)) receptor (TP). The aim of this study was to evaluate the pharmacological profile of inhaled KP-496 and its effects on airway obstruction. EXPERIMENTAL APPROACH: Antagonist activities of inhaled KP-496 were investigated using bronchoconstriction induced in guinea pigs by LTD(4) or U46619, a stable TXA(2) mimetic. Guinea pigs sensitized with injections of ovalbumin were used to assess the effects of inhaled KP-496 on bronchoconstriction induced by antigen (i.v.). Another set of guinea pigs were sensitized and challenged with ovalbumin by inhalation and the effects of inhaled KP-496 on immediate and late airway responses and airway hyperresponsiveness were investigated. KEY RESULTS: KP-496 significantly inhibited LTD(4)- and U46619-induced bronchoconstriction in a dose-dependent manner. The inhibitory effects of KP-496 (1%) were comparable to those of montelukast (a CysLT(1) antagonist, p.o., 0.3 mg kg(-1)) or seratrodast (a TP antagonist, p.o., 3 mg kg(-1)). KP-496 (1%) and oral co-administration of montelukast (10 mg kg(-1)) and seratrodast (20 mg kg(-1)) significantly inhibited antigen-induced bronchoconstriction, whereas administration of montelukast or seratrodast separately did not inhibit antigen-induced bronchoconstriction. KP-496 exhibited dose-dependent and significant inhibitory effects on the immediate and late airway responses and airway hyperresponsiveness following antigen challenge. CONCLUSIONS AND IMPLICATIONS: KP-496 exerts effects in guinea pigs which could be beneficial in asthma. These effects of KP-496 were greater than those of a CysLT(1) antagonist or a TP antagonist, in preventing antigen-induced airway obstruction.

Human normal hepatocytes are susceptible to apoptosis signal mediated by both TRAIL-R1 and TRAIL-R2.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) triggers apoptosis in tumor cells without toxicity to normal cells, but some recombinant versions of TRAIL caused hepatocyte death. We generated fully human monoclonal antibodies (mAbs) that bind specifically to TRAIL receptor 1 (TRAIL-R1) and TRAIL receptor 2 (TRAIL-R2), which mediate apoptosis signal when they ligate with TRAIL, to investigate the contribution of each receptor to induce tumor cell apoptosis and hepatocyte toxicity. All of mAbs to TRAIL-R1 and TRAIL-R2 induced cell death in several cancer cell lines susceptible to TRAIL but not in human umbilical vein endothelial cells in vitro. Both anti-TRAIL-R1 mAbs and anti-TRAIL-R2mAbs also caused cell death in hepatocytes. However, a subset of mAbs to TRAIL-R2, which was characterized by the TRAIL blocking activity, did not show strong hepatocyte toxicity. These results indicate that human normal hepatocytes are susceptible to both TRAIL-R1- and TRAIL-R2-mediated apoptosis signal. Cell Death and Differentiation (2004) 11, 203-207. doi:10.1038/sj.cdd.4401331 Published online 24 October 2003

Gastric restitution is inhibited by dexamethasone, which is reversed by hepatocyte growth factor and rebamipide.

BACKGROUND: Glucocorticoids have been shown to induce peptic ulcers, especially when co-administered with NSAIDs. Hepatocyte growth factor (HGF) plays a role in gastric ulcer repair, facilitating the restitution of gastric mucosal epithelial cells. HGF expression is induced by PGs in gastric fibroblasts. We hypothesized that dexamethasone (DEX) may inhibit PG production and HGF expression, thus inhibiting HGF-induced gastric epithelial restitution. AIM: To investigate the effect of DEX on gastric restitution, using cultured gastric cells, the role of HGF in the restitution inhibited by DEX, and the effect of rebamipide on DEX- inhibited restitution. METHODS: Human gastric fibroblasts were prepared from human stomach obtained at surgery; PGE2 and HGF is determined by ELISA; Restitution was assessed by the round wound restitution model, using coculture of gastric fibroblasts and epithelial cells; COX-2 and HGF mRNA were quantified by TaqMan RT-PCR system. RESULTS: 1. DEX inhibited HGF mRNA and COX-2 mRNA. Accordingly, it inhibited PGE2 and HGF release. 2. DEX inhibited the restitution of gastric cells. 3. The inhibition of restitution was reversed by HGF and rebamipide to the same extent. 4. Rebamipide induced PGE2 and HGF. CONCLUSION: DEX inhibits restitution via HGF depletion, and rebamipide reverses the inhibited restitution by HGF induction.

Epidermoid cyst originating from an intrapancreatic accessory spleen.

Pancreatectomy in a 45-year-old woman showed, after pathologic analysis, an epidermoid cyst originating from an intrapancreatic accessory spleen. This mass consisted of parenchymal and cystic components. It is important to note the relation between the splenic parenchyma and the parenchymal component of the mass for the differential diagnosis of a cystic mass in the pancreatic tail.

[Spontaneous rupture of renal angiomyolipoma with tuberous sclerosis during long-term follow-up: a case report].

We present a case of spontaneous rupture of bilateral renal angiomyolipoma (AML) with tuberous sclerosis. A 46-year-old woman was admitted with sudden onset of severe left flank pain. She had been diagnosed to have bilateral AML with tuberous sclerosis 15 years earlier. Four years after the initial diagnosis, spontaneous rupture of right renal AML occurred and right renal embolization of the right renal artery was performed. The treatment for the left renal AML was not performed. Eleven years later in 2000, spontaneous rupture of contralateral renal AML occurred and left renal embolization of the left renal artery was performed. We evaluated the efficacy of selective arterial embolization in right AML and the change of the tumor size during a 10-year follow up after embolization. The right AML had decreased 86% in 11 years. Selective arterial embolization is an effective and safe treatment for AML. We evaluated the natural history of left AML and calculated the doubling time to be about 1,370 days for the first period of 4 years and about 2,075 days for the second period of 11 years. Although the growth change was very slow, we should observe the tumors carefully on computed tomography or ultrasound to prevent life-threatening hemorrhage.

Direct interaction of insulin-like growth factor-1 receptor with leukemia-associated RhoGEF.

Insulin-like growth factor (IGF)-1 plays crucial roles in growth control and rearrangements of the cytoskeleton. IGF-1 binds to the IGF-1 receptor and thereby induces the autophosphorylation of this receptor at its tyrosine residues. The phosphorylation of the IGF-1 receptor is thought to initiate a cascade of events. Although various signaling molecules have been identified, they appear to interact with the tyrosine-phosphorylated IGF-1 receptor. Here, we identified leukemia-associated Rho guanine nucleotide exchange factor (GEF) (LARG), which contains the PSD-95/Dlg/ZO-1 (PDZ), regulator of G protein signaling (RGS), Dbl homology, and pleckstrin homology domains, as a nonphosphorylated IGF-1 receptor-interacting molecule. LARG formed a complex with the IGF-1 receptor in vivo, and the PDZ domain of LARG interacted directly with the COOH-terminal domain of IGF-1 receptor in vitro. LARG had an exchange activity for Rho in vitro and induced the formation of stress fibers in NIH 3T3 fibroblasts. When MDCKII epithelial cells were treated with IGF-1, Rho and its effector Rho-associated kinase (Rho-kinase) were activated and actin stress fibers were enhanced. Furthermore, the IGF-1-induced Rho-kinase activation and the enhancement of stress fibers were inhibited by ectopic expression of the PDZ and RGS domains of LARG. Taken together, these results indicate that IGF-1 activates the Rho/Rho-kinase pathway via a LARG/IGF-1 receptor complex and thereby regulates cytoskeletal rearrangements.

Differentiation stages of eosinophils characterized by hyaluronic acid binding via CD44 and responsiveness to stimuli.

To characterize interleukin (IL)-5-induced eosinophils, we examined the expression of CD44, very late antigen (VLA)-4, and the IL-5 receptor alpha chain, as well as the levels of eosinophil peroxidase and the generation of superoxide. Eosinophils were prepared from IL-5-transgenic mice, then characterized using electron microscopy to determine their responses to stimuli. Whereas CD44 densities remained almost constant, the level of VLA-4 increased in parallel with eosinophil maturation. Although a subset of IL-5-induced eosinophils with high side scatter recovered from bone marrow and rare ones found in blood recognized hyaluronic acid (HA), most did not have this property. Bone marrow eosinophils with high side scatter and lower density contained eosinophil peroxidase, not only in granules, but also in membranous structures for 30% of this population. This population developed HA-binding ability in response to IL-3, IL-4, IL-5, granulocyte-macrophage colony-stimulating factor, macrophage inflammatory protein (MIP)-2, monocyte chemotactic protein (MCP)-1, eotaxin, nerve growth factor (NGF), and opsonized zymosan (OZ). Peripheral blood eosinophils acquired HA-binding ability in response to the same stimuli, but their responses were less than those of bone marrow eosinophils with high levels of side scatter. However, splenic eosinophils did not respond to these stimuli. Although peripheral blood eosinophils did not proliferate when stimulated by IL-5, these were the only cells that released eosinophil peroxidase in response to IL-4, MIP-2, MCP-1, eotaxin, NGF, and OZ. With the exception of a subset of bone marrow eosinophils, the ability to acquire HA binding, but not the ability to generate superoxide, correlated with eosinophil peroxidase activity and major basic protein accumulation in the granules of maturing cells.

Cancer regression induced by modified CTL therapy is regulated by HLA class II and class I antigens in Japanese patients with advanced cancer.

Autologous cancer-specific bulk CTLs are unlikely to be induced by in vitro CTL generation (ivtCTLG) using peripheral blood mononuclear cells (PBMCs) of cancer patients when autologous cancer cells are used as in vitro stimulators. However, autologous cancer-specific bulk CTLs are frequently activated when allogeneic cancer cells are used as in vitro stimulators, regardless of the type of cancer cell. We have developed a cancer-specific immunotherapy called modified CTL therapy, which involves adoptive immunotherapy of autologous cancer-specific bulk CTLs after active immunization of autologous or allogeneic cancer cells screened as in vitro stimulators according to their ability to induce autologous cancer-specific CTLs (ACS. CTLs). Cancer did not regress in patients in whom ACS.CTLs were not induced by ivtCTLG using the patients' PBMCs in therapy. Cancer regression, albeit temporary, occurred solely in patients under the immunological condition that ACS.CTLs were induced by ivtCTLG using PBMCs through the therapy. The induction of ACS.CTLs by ivtCTLG using patient PBMCs in therapy was related to patients' HLA class II antigens. HLA DR8 was seen more frequently in ACS.CTL-inducible patients than in ACS.CTL-uninducible patients (P=0.051). On the contrary, HLA DQ3 was seen more frequently in ACS.CTL-uninducible patients (P=0.055). On the other hand, the success in therapy, albeit temporary, was related mainly to patients' HLA class I antigens. HLA B61 was seen more frequently in patients whose therapy proved effective than in patients whose therapy proved ineffective (P=0.018). HLA Cw7 was seen more frequently in therapy-ineffective patients (P=0.040).

Safety evaluation of yeast glutaminase.

The consumption of soy and soy products (including soy sauce) has been increasing in Western countries due to purported health benefits of soy (cancer protective, estrogenic effects). In addition to providing soy proteins and isoflavones, soy sauce also functions as a flavor enhancer and is able to impart a "umami" taste. Glutaminases are used in the production of soy sauce and enzymatically hydrolyzed protein. The glutaminases described herein were produced from the cultured broth of Cryptococcus albidus (ATCC-20293) which is designated as CK, a mutant of C. albidus (ATCC-20293) which is designated as CK-D10 and the newly isolated Cryptococcus sp. NISL-3771 which is designated as TK. All three preparations (CK, CK-D10 and TK) were evaluated for pathogenicity and virulence in mice and were found to be non-pathogenic. The acute LD(50)s for CK in male mice was greater than 4.8 g/kg body weight and for female mice was greater than 6.5 g/kg body weight. Acute LD(50)s for CK and CK-D10 in male and female rats was greater than 7.5 g/kg body weight, and that for TK was greater than 10 g/kg body weight. Subchronic (90-day) feeding studies (wherein the glutaminases were presented as dietary admixtures) were conducted in mice and rats. The NOAEL for CK in mice was 7.5 g/kg body weight/day. The NOAELs in rats were as follows: for CK, 9 g/kg body weight/day; for CK-D10, 1.2 g/kg body weight/day, and for TK, 8 g/kg body weight/day. Mice received CK as a dietary admixture at levels of 0, 1.0 and 10.0% for 1 year. The NOAEL was 13 g/kg body weight/day. The glutaminases from C. albidus described herein demonstrate very low toxicity.

Prevention of colonic aberrant crypt foci by dietary feeding of garcinol in male F344 rats.

The modifying effects of dietary feeding of a polyisoprenylated benzophenone, garcinol, isolated from Garcinia indica fruit rind on the development of azoxymethane (AOM)-induced colonic aberrant crypt foci (ACF) were investigated in male F344 rats. We also assessed the effects of garcinol on proliferating cell nuclear antigen (PCNA) index in ACF and activities of detoxifying enzymes of glutathione S-transferase (GST) and quinone reductase (QR) in liver. In addition, we examined the effects of garcinol on 12-O-tetradecanoylphorbol-13-acetate-induced O(2)(-) generation in differentiated human promyelocytic HL-60 cells and lipopolysaccharide (LPS)- and interferon (IFN)-gamma-induced nitric oxide (NO) generation in mouse macrophage RAW 264.7 cells. Western blotting analysis of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expression was done in LPS- and IFN-gamma-treated mouse macrophage RAW 264.7 cells. Rats were given subcutaneous injections of AOM (15 mg/kg body wt) once a week for 3 weeks to induce ACF. They also received the experimental diet containing 0.01 or 0.05% garcinol for 5 weeks, starting 1 week before the first dosing of AOM. AOM exposure produced 97 +/- 15 ACF/rat at the end of the study (week 5). Dietary administration of garcinol caused significant reduction in the frequency of ACF: 72 +/- 15 (26% reduction, P < 0.01) at a dose of 0.01% and 58 +/- 8 (40% reduction, P < 0.001) at a dose of 0.05%. Garcinol administration significantly lowered PCNA index in ACF. Feeding of garcinol significantly elevated liver GST and QR activities. In addition, garcinol could suppress O(2)(-) and NO generation and expression of iNOS and COX-2 proteins. These findings might suggest possible chemopreventive ability of garcinol, through induction of liver GST and QR, inhibition of O(2)(-) and NO generation and/or suppression of iNOS and COX-2 expression, on colon tumorigenesis.

[Successful treatment of a patient with recurrent ovarian clear cell adenocarcinoma under combination chemotherapy of 5-FU (civ) and low-dose CDDP (i.v.)].

Resistance to conventional chemotherapy including CDDP is the most important therapeutic problem in ovarian cancer. The combination chemotherapy of 5-FU (civ) and low-dose CDDP (i.v.) was applied to a patient with recurrent ovarian clear cell adenocarcinoma (stage IIa), which is often more resistant to systemic chemotherapy than other ovarian adenocarcinomas and is a poor prognostic factor. The patient underwent cytoreductive surgery. Then, 5-FU 375 mg/m2/day civ (days 1-5, 8-12, 15-19, 22-26) and CDDP 3.75 mg/m2/day i.v. (days 1-5, 8-12, 15-19, 22-26) were administered. After four courses of this treatment, there is no sign of recurrence. This result indicates that the combination of 5-FU and CDDP is useful in the treatment of recurrent ovarian cancers.