Insulinoma induces a hyperinsulinemia-mediated decrease of GLUT2 and GLP1 receptor in normal pancreatic ß-cells.

There have been several clinical reports of transient postoperative hyperglycemia in patients with insulinoma, but the effect of insulinoma on normal ß-cells has not been investigated. We examined the glucose transporter 2 (GLUT2) and glucagon-like peptide 1 receptor (GLP1R) expression in normal pancreatic ß-cells of five patients with insulinoma and five patients with normal glucose tolerance (NGT) as controls. The positive rate of GLUT2-or GLP1R-positive islets in the nontumor area was calculated by the ratio with the analyzed islets. For functional in vitro analyses, q-PCR and Western blotting were performed after insulin loading on MIN6 cells. The expression rates of both GLUT2 and GLP1R were significantly lower in nontumor area islets of insulinoma patients than in patients with NGT (GLUT2: 31.6 ± 15.3% vs 95.9 ± 6.7%, p < 0.01, GLP1R: 66.8 ± 15.0% vs 96.7 ± 5.0%, p < 0.01). Exposure of MIN6 cells to high concentrations of insulin resulted in a significant decrease in GLUT2 protein for 12 h and GLP1R protein for 24 h (GLUT2; 1.00 ± 0.079 vs 0.81 ± 0.04. p = 0.02, GLP1R; 1.00 ± 0.10 vs 0.50 ± 0.24, p = 0.03) but not in those mRNAs. Our findings show that insulinoma is associated with the downregulation of GLUT2 and GLP1R expression in nontumor area islets. These phenomena may be caused by high levels of insulin.

Effects of short-chain fatty acids on Actinomyces naeslundii biofilm formation.

Actinomyces naeslundii is an early colonizer and has important roles in the development of the oral biofilm. Short-chain fatty acids (SCFA) are secreted extracellularly as a product of metabolism by gram-negative anaerobes, e.g. Porphyromonas gingivalis and Fusobacterium nucleatum; and the SCFA may affect biofilm development with interaction between A. naeslundii and gram-negative bacteria. Our aim was to investigate the effects of SCFA on biofilm formation by A. naeslundii and to determine the mechanism. We used the biofilm formation assay in 96-well microtiter plates in tryptic soy broth without dextrose and with 0.25% sucrose using safranin stain of the biofilm monitoring 492 nm absorbance. To determine the mechanism by SCFA, the production of chaperones and stress-response proteins (GrpE and GroEL) in biofilm formation was examined using Western blot fluorescence activity with GrpE and GroEL antibodies. Adding butyric acid (6.25 mm) 0, 6 and 10 h after beginning culture significantly increased biofilm formation by A. naeslundii, and upregulation was observed at 16 h. Upregulation was also observed using appropriate concentrations of other SCFA. In the upregulated biofilm, production of GrpE and GroEL was higher where membrane-damaged or dead cells were also observed. The upregulated biofilm was significantly reduced by addition of anti-GroEL antibody. The data suggest biofilm formation by A. naeslundii was upregulated dependent on the production of stress proteins, and addition of SCFA increased membrane-damaged or dead cells. Production of GroEL may physically play an important role in biofilm development.

A triploid partial mole placenta from paternal isodisomy with a diploid fetus derived from one sperm and one oocyte may have caused angiogenic imbalance leading to preeclampsia-like symptoms at 19 weeks of gestation.

Partial hydatidiform mole with a normal fetus is extremely rare. A 30-year-old woman presented at 19 weeks gestation with clinical manifestations of severe preeclampsia. The fetus revealed a normal 46,XX karyotype and the placenta revealed triploid 69,XXX from paternal isodisomy. Microsatellite analysis revealed that the fetus and the triploid partial mole were derived from one sperm and one oocyte, followed by duplication of paternal chromosomes in only a trophectodermal cell. The maternal serum levels of angiogenic factors were extremely high compared with those reported in preeclampsia, suggesting an angiogenic imbalance may have caused preeclampsia-like symptoms before 20 weeks of gestation.

Predominance of ß-cell neogenesis rather than replication in humans with an impaired glucose tolerance and newly diagnosed diabetes.

CONTEXT: A decrease in pancreatic ß-cell mass is involved in the development of type 2 diabetes. OBJECTIVE: The purpose of this study was to evaluate the ß-cell mass and the incidence of ß-cell neogenesis, replication, and apoptosis at both the prediabetic and diabetic stages. METHODS: We conducted a cross-sectional study of pancreatic tissues obtained from 42 patients undergoing a pancreatectomy who were classified into 4 groups: normal glucose tolerance (n = 11), impaired glucose tolerance (n = 11), newly diagnosed diabetes (n = 10), and long-standing type 2 diabetes (n = 10). RESULTS: The relative ß-cell area decreased and the ß-cell apoptosis increased during the development of diabetes. The number of single and clustered ß-cells, some of which coexpressed nestin, increased in the patients with impaired glucose tolerance and newly diagnosed diabetes. The prevalence of cells positive for both insulin and glucagon or somatostatin also increased in these patients compared with those with normal glucose tolerance. These double-positive cells were mainly localized in single and clustered ß-cells, rather than large islets, and were also positive for Pdx1 or Ngn3. The percentage of insulin-positive cells embedded within ducts increased in the impaired glucose tolerance group. There were no significant differences in the incidence of cells positive for both insulin and Ki67 among the groups. CONCLUSIONS: These results suggest that ß-cell neogenesis, rather than replication, predominates during impaired glucose tolerance and newly diagnosed diabetes in humans and may serve as a compensatory mechanism for the decreased ß-cell mass.

Assessment of the plasma/serum IgG test to screen for periodontitis.

Chronic periodontitis is a silent infectious disease prevalent worldwide and affects lifestyle-related diseases. Therefore, efficient screening of patients is essential for general health. This study was performed to evaluate prospectively the diagnostic utility of a blood IgG antibody titer test against periodontal pathogens. Oral examination was performed, and IgG titers against periodontal pathogens were measured by ELISA in 1,387 individuals. The cut-off value of the IgG titer was determined in receiver operating characteristic curve analysis, and changes in periodontal clinical parameters and IgG titers by periodontal treatment were evaluated. The relationships between IgG titers and severity of periodontitis were analyzed. The best cut-off value of IgG titer against Porphyromonas gingivalis for screening periodontitis was 1.682. Both clinical parameters and IgG titers decreased significantly under periodontal treatment. IgG titers of periodontitis patients were significantly higher than those of healthy controls, especially in those with sites of probing pocket depth over 4 mm. Multiplied cut-off values were useful to select patients with severe periodontitis. A blood IgG antibody titer test for Porphyromonas gingivalis is useful to screen hitherto chronic periodontitis patients.

Topical and intermittent application of parathyroid hormone recovers alveolar bone loss in rat experimental periodontitis.

BACKGROUND AND OBJECTIVE: Periodontitis is characterized by periodontal tissue inflammation and alveolar bone loss. The intermittent administration of parathyroid hormone (PTH), a major regulator of bone remodeling, has been demonstrated to stimulate osteoblastic activity. Although the systemic administration of PTH has been reported to protect against periodontitis-associated bone loss, the effect of the topical administration of PTH is unclear. In this study, the effect of intermittent administration of PTH on osteoblastic differentiation was examined in cultured calvaria cells and then the effect of topical and intermittent administration of PTH was determined by measuring the recovery of alveolar bone loss after inducing experimental periodontitis in rats. MATERIAL AND METHODS: Alkaline phosphatase activity and bone nodule formation were measured in fetal rat calvaria cells. Experimental periodontitis was induced by placing nylon ligature around rat maxillary molars for 20 d. After ligature removal (day 0), PTH was topically injected into buccal gingiva three times a week for 10 wk. Micro-computed tomography analysis and histological examination were performed on days 35 and 70. RESULTS: Intermittent exposure of PTH in calvaria cells increased alkaline phosphatase activity and bone nodule formation by 1.4- and 2.4-fold, respectively. Ligature procedures induced marked alveolar bone loss around the molars on day 0 and greater bone recovery was observed in the PTH-treated rats on day 70. An increase in osteoid formation on the surface of alveolar bone was detected in the PTH-treated rats. CONCLUSION: Intermittent treatment with PTH stimulated osteoblastic differentiation in fetal rat calvaria cell cultures, and topical and intermittent administration of PTH recovered alveolar bone loss in rat experimental periodontitis.

Wound healing effects of gingival fibroblasts cultured in animal-free medium.

OBJECTIVE: The purpose of this study was to develop a graft material made of gingival fibroblasts cultured in animal-free medium (HFDM1). METHODS: We examined the effects of human serum (HS) on cell growth and wound healing capability, demonstrated by cytokine production, of gingival fibroblasts cultured in HFDM1. Subsequently, the capability of fibroblasts cultured in HFDM1 with 2% HS to promote the healing of skin defects was evaluated using nude mice. RESULTS: The proliferation of human gingival fibroblasts was increased when HS at a concentration of 0.5-2% was added to HFDM1. Wound healing cytokines, including transforming growth factor-beta, keratinocyte growth factor, hepatocyte growth factor, vascular endothelial growth factor, and IL-6 produced by gingival fibroblasts were increased by adding 2% HS to HFDM1. In addition, gingival fibroblasts cultured in HFDM1 with 2% HS improved wound healing of mouse skin defects as well as those cultured in Dulbecco's modified Eagle's medium with 10% fetal calf serum. CONCLUSION: Gingival fibroblasts cultured in HFDM1 with 2% HS may be useful as a graft material for reconstruction.

Inhibiting effects of Streptococcus salivarius on competence-stimulating peptide-dependent biofilm formation by Streptococcus mutans.

INTRODUCTION: The effects of Streptococcus salivarius on the competence-stimulating peptide (CSP)-dependent biofilm formation by Streptococcus mutans were investigated. METHODS: Biofilms were grown on 96-well microtiter plates coated with salivary components in tryptic soy broth without dextrose supplemented with 0.25% sucrose. Biofilm formations were stained using safranin and quantification of stained biofilms was performed by measuring absorbance at 492 nm. RESULTS: S. mutans formed substantial biofilms, whereas biofilms of S. salivarius were formed poorly in the medium conditions used. Furthermore, in combination cultures, S. salivarius strongly inhibited biofilm formation when cultured with S. mutans. This inhibition occurred in the early phase of biofilm formation and was dependent on inactivation of the CSP of S. mutans, which is associated with competence, biofilm formation, and antimicrobial activity of the bacterium, and is induced by expression of the comC gene. Comparisons between the S. mutans clinical strains FSC-3 and FSC-3DeltaglrA in separate dual-species cultures with S. salivarius indicated that the presence of the bacitracin transport ATP-binding protein gene glrA caused susceptibility to inhibition of S. mutans biofilm formation by S. salivarius, and was also associated with the regulation of CSP production by com gene-dependent quorum sensing systems. CONCLUSION: It is considered that regulation of CSP by glrA in S. mutans and CSP inactivation by S. salivarius are important functions for cell-to-cell communication between biofilm bacteria and oral streptococci such as S. salivarius. Our results provide useful information for understanding the ecosystem of oral streptococcal biofilms, as well as the competition between and coexistence of multiple species in the oral cavity.

Confinement of a large number of antiprotons and production of an ultraslow antiproton beam.

We have used a radio frequency quadrupole decelerator to decelerate antiprotons emerging from the CERN Antiproton Decelerator from MeV- to keV-scale energy, and collected five decelerated pulses in a multiring trap. Some 5 x 10(6) antiprotons were stacked in this way. Cooling of the trapped antiprotons by a simultaneously trapped electron plasma was studied nondestructively via shifts in plasma mode frequencies. We have also demonstrated the first step in extracting a 10-500 eV antiproton beam from the trap.

Breath alcohol concentrations in Japanese outpatients following paclitaxel and docetaxel infusion.

Patients receiving paclitaxel or docetaxel also receive a significant amount of ethanol, as both products contain ethanol as solvent. Patients in our clinics have occasionally exhibited signs of alcohol intoxication immediately after paclitaxel infusion. In 2002, the Japanese government lowered the minimum ethanol concentration for the definition of drunk driving, with the threshold breath alcohol concentration (BRAC) of 0.15 mg/l. The aim of this study was to measure BRAC in Japanese outpatients treated with paclitaxel or docetaxel and to assess intoxication according to this standard. Fifty-two Japanese patients were enrolled from October 2003 to February 2004. Patient characteristics were as follows: male/female, 13/39: median age, 71 (range: 34-78); breast/lung/ovarian cancer 24/16/12; and paclitaxel/docetaxel treatment: 36/16, respectively. The mean total doses of paclitaxel or docetaxel were 178 mg (range: 107-300) and 53 mg (30-100), respectively. Breath samples were measured three times immediately following the infusion of paclitaxel or docetaxel via ethyl alcohol detector and the mean value was recorded. BRAC was detected in 20 patients (56%) with paclitaxel and in none of the docetaxel patients. BRAC was measured again 30 min after the initial measurement in BRAC-detected cases with the patients' permission. In four of six BRAC-remeasured patients, BRAC became undetectable after 30 min. There was no correlation between the total doses of paclitaxel and BRAC or between the infusion rates of paclitaxel and BRAC. In conclusion, clinicians should recognize the potential for alcohol intoxication with paclitaxel administration. Patients should be instructed to avoid driving on the day of paclitaxel administration.

Results of surgical treatment for non-small cell lung cancer of 20 mm or less in diameter.

BACKGROUND: Surgical efficacy is still unsatisfactory for small lung cancer; accordingly, minimal resection has recently been the focus of increased study. The objective of the current study was to evaluate the factors associated with small lung cancer, and to determine whether such factors are reliable predictors of long-term survival. METHODS: We retrospectively investigated 130 patients with histologically confirmed non-small cell carcinoma, whose treatments were primarily surgical, with no chemotherapy or radiotherapy prior to surgery. All tumors were located peripherally and were less than 20 mm in diameter. Follow-up was performed for five-year and eight-year survivors and multivariate analysis with Cox's proportional hazards regression model was performed. RESULTS: Of all 130 patients, the 5-year survival rate among patients with tumors less than 15 mm was 82.5 %, vs. 57.4 % of patients with tumors with a diameter of 16 - 20 mm. The 5-year survival rate of patients who were node negative was 73.9 % while it was 28.5 % for node-positive patients. Status of nodal invasion was also significantly associated with survival in small-size tumors ( p < 0.0001). Furthermore, the 5-year survival rate among patients with pleural involvement was 55 % vs. 83.8 % for patients without pleural involvement. Using multivariate Cox analysis, lymph node involvement ( p = 0.0004), size ( p = 0.0475), and pleural invasion ( p = 0.0482) were found to be independent prognostic factors in cases of tumors 20 mm or less in diameter. CONCLUSIONS: The results of this study at least demonstrate that the optimal therapy for patients with nodal involvement or patients with tumors of 16 - 20 mm must be carefully determined even in cases of small lung cancer.

Pacemaker activity from submucosal interstitial cells of Cajal drives high-frequency and low-amplitude circular muscle contractions in the mouse proximal colon.

The present study was aimed at elucidating how pacemaker activity (plateau potentials) (mean frequency: 15.9 +/- 2.8 times min(-1)) from submucosal interstitial cells of Cajal (ICC-SM) control spontaneous contractions in the mouse proximal colon. Mechanical activities in the circular muscle direction showed high-frequency (mean frequency: 15.6 +/- 2.7 times min(-1)) and low-amplitude (mean amplitude: 0.01 +/- 0.005 g) (HFLA) rhythmic contractions. Simultaneous recordings of circular muscle mechanical activity and electrical activity from ICC-SM revealed that HFLA contractions were synchronized with plateau potentials (mean frequency: 15.9 +/- 2.8 times min(-1)). Although low-frequency (3.5 +/- 2.1 times min(-1)) and high-amplitude (0.12 +/- 0.03 g) (LFHA) contractions in both longitudinal and circular muscle directions were synchronized with burst of action potentials in both muscle cells, these LFHA contractions were not synchronous with plateau potentials. Intracellular Ca2+ release from the internal stores through IP3 receptors is not a major factor to generate both action potentials differently from plateau potentials. Neither tetrodotoxin nor atropine affected the plateau potentials. The results reveal that the pacemaker activity from ICC-SM drives only the spontaneous HFLA (one-tenth amplitude of the LFHA circular and longitudinal muscle contractions) circular muscle contractions without control by enteric nerves.

Serum granulysin is a marker for Th1 type immunity in pre-eclampsia.

Recent studies suggest that pre-eclampsia is associated with a Th1 predominant state and may be considered a failure of tolerance. Granulysin is a cytotoxic granule protein of natural killer (NK) cells and cytotoxic T lymphocytes (CTLs). Recently, we developed an enzyme-linked immunosorbent assay (ELISA) system for detecting serum granulysin, and reported that serum granulysin is a useful marker to evaluate the cell-mediated immunity. In this study, we show that the serum levels of granulysin were significantly elevated in pre-eclamptic patients compared with those in normal pregnancy subjects. In addition, the serum granulysin levels in pre-eclamptic patients were well associated with mean blood pressure, percentage of peripheral blood Th1 cells and Th1/Th2 ratios. The present results suggest that the serum granulysin levels would be a useful and novel serum marker to evaluate the Th1/Th2 balance, especially Th1 type immunity in pre-eclampsia.

Brain lesions in the course of generalised tetanus.

A 47-year-old woman developed left hemiparesis primarily affecting the lower limbs during the course of severe generalised tetanus. MRI on the 82nd hospital day revealed cortical and subcortical lesions predominantly in the right frontal and parietal lobes in addition to marked brain atrophy. Three months later, the enhancing lesions were still present on follow up MRI. We postulate that structures above brainstem may be involved in severe generalised tetanus.

Clinicopathological analysis of 143 primary malignant lymphomas in the small and large intestines based on the new WHO classification.

AIM: To study the clinicopathological and immunohistochemical features of 143 cases of primary small and large intestinal non-Hodgkin's lymphoma (NHL) in Japanese patients who presented between 1981 and 2000. METHODS AND RESULTS: The new World Health Organization (WHO) classification was used to classify NHL. The patients included 109 males and 34 females, with an average age of 54.1 years. Tumour sites were as follows: ileocaecal (n = 51, 35.7%), ileum (n = 29, 20.3%), rectum (n = 13, 9.1%), and duodenum (n = 11, 7.7%). Macroscopically, 124 cases (86.7%) were classified as tumorous type, 12 (8.4%) as diffuse infiltration type (erosion, superficial ulceration), five (3.5%) as polyposis type, and only two cases (1.4%) as ulceration type. Immunohistochemically, 122 lesions (85.3%) were of B-cell phenotype and 21 lesions (14.7%) were of T-cell phenotype. According to the WHO classification, of the B-cell lymphomas, 84 cases (68.9%) were large cell, 16 (13.1%) were Burkitt, 10 (8.2%) were marginal zone B-cell lymphomas of mucosa-associated lymphoid tissue (MALT), and seven (5.7%) were mantle cell tumours. Among the T-cell lymphomas, 15 (71.4%) were of unspecified type, two (9.5%) were natural killer type, two were anaplastic large-cell lymphomas, one was lymphoblastic, and one was an adult T-cell leukaemia lymphoma. The survival rate for T-cell lymphomas was poorer than for B-cell lymphomas. Among the B-cell lymphomas, mantle cell lymphoma tended to have a poorer prognosis, whereas MALT lymphomas had a better prognosis than other B-cell tumour types. CONCLUSIONS: Our retrospective study of patients with primary malignant lymphomas in the small and large intestines has illustrated the clinical features and outcomes of patients with this disease.

Video-assisted thoracic needle aspiration cytology for malignancy of the peripheral lung.

BACKGROUND: The technique of video-assisted thoracic fine-needle aspiration cytology (VAT-FNA) to the lung has been described in very few publications, and its diagnostic role has not been evaluated so far. Thus current studies focus on whether the diagnostic role could be applied usefully to VAT-FNA of peripheral lung lesions. METHODS: Between January 1995 and January 2000, a total of one hundred and twenty-eight cases of VAT-FNA on lung tumors were reviewed retrospectively. The superficial lung was visualized a part of directly or indirectly by scope and a 22-gauge needle inserted for FNA. Material was expressed onto glass slides, and smears were stained by our modified quick-stain method. The cytological diagnoses based on VAT-FNA were reviewed and compared with the final histopathological diagnoses. RESULTS: The cytological diagnosis was true positive in 100 cases (93.5% sensitivity), whereas the true negative result in 20 cases was 95.2% specificity. The false-positive rate was 4.8%, and false-negative results were 6.5%. The accordance in all malignant cases between cytology and histology was 73.8%. VAT-FNA caused no difficulties in any of the cases. CONCLUSION: The application of VAT-FNA to the peripheral lung lesion is not only useful, cost-beneficial, safe and minimally invasive but also accurate. Especially, this method may play an important role in cases of suspected malignancy in peripheral cases.

[Surgical therapy for patients with thymoma or thymic carcinoma; based on WHO classification].

The treatment strategy for patients with thymoma or thymic carcinoma still has not been confirmed, and the molecular biologic characteristics of these tumors have not been defined also. We have emphasized the clinical significance of this classification so far by the research cooperation with professor Müller-Hermelink, the institute of pathology, Würzburg University. This time, We introduced clinico-pathological findings based on the new WHO classification in 59 Case of thymic epithelial tumor and reffered the treatment strategy for patients with thymic cancer.

Enhancement of osteogenesis on hydroxyapatite surface coated with synthetic peptide (EEEEEEEPRGDT) in vitro.

Some dental implants are coated with hydroxyapatite (HA), which preferentially binds to bone. Several matrix proteins have an arginine-glycine-aspartic acid (RGD) sequence where cells attach via an integrin receptor. We hypothesized that coating an HA surface with an RGD-containing peptide might enhance the attachment and differentiation of osteoblasts. The HA disks (diameter 34 mm, thickness 1 mm) were treated with a solution (50 mM Tris/HCl and 150 mM NaCl, pH 7.4) containing the peptide EEEEEEEPRGDT, in which the E repetition exerts a high affinity to HA. After washing with phosphate-buffered saline, KUSA/A1 mouse osteoblastic cells were inoculated onto the HA surface and cultured. After 30 min, the number of cells attached to the surface was counted. The DNA content and alkaline phosphatase (ALP) activity were measured after 10 days in culture. Expression of bone matrix proteins was also examined by means of reverse transcriptase-polymerase chain reaction at 7 days; the mineralized area of the culture was also evaluated by staining with Alizarin Red S after 10 days. Treatment with the peptide stimulated cell attachment and increased DNA content and ALP activity. Furthermore, matrix protein expression and mineralized nodule formation were enhanced to a greater extent on the peptide-treated surface than on the nontreated surface. Our results indicate that coating an HA surface with RGD-containing peptide enhances osteoblast attachment and differentiation. This peptide treatment of HA-coated implants may stimulate the osseointegration of the implants.

Biosynthesis of versican by rat dental pulp cells in culture.

The biosynthesis of proteoglycans by these cultured pulp cells was investigated by metabolic labelling, using [(35)S]sulphate, [(3)H]glucosamine and [(3)H]leucine as precursors. Versican-like large proteoglycan, decorin- and biglycan-like small proteoglycans and a small amount of sulphated protein were released into the culture medium. Heparan sulphate species were also identified in cell-layer extracts. Versican-like proteoglycan had an average molecular mass of approximately 800kDa. The molecular mass of chondroihnase ABC-digested core protein exhibited heterogeneity, ranging from 250 to 400kDa, and the glycosaminoglycan chains had an average molecular mass of approximately 42kDa. These results indicate the presence of 10-13 glycosaminoglycan chains per core protein, consistent with the characteristics of versican. This glycosaminoglycan chain contained approximately 63% 4-sulphated disaccharides.