Characterisation of a sucrose-independent in vitro biofilm model of supragingival plaque.

OBJECTIVES: Sugar consumption has been decreasing in Japan, suggesting higher rates of sucrose-independent supragingival plaque formation. For developing an in vitro biofilm model of sucrose-independent supragingival plaque, this study aimed to investigate the compositions and functions on contributing to cariogenicity in comparison with sucrose-dependent biofilm. MATERIALS AND METHODS: An in vitro multispecies biofilm containing Actinomyces naeslundii, Streptococcus gordonii, S. mutans, Veillonella parvula and Fusobacterium nucleatum was formed on 24-well plates in the absence or presence of 1% sucrose. Compositions were assessed by plate culture, scanning electron microscopy and confocal laser scanning microscopy after fluorescent in situ hybridisation or labelling of extracellular polymeric substances (EPS). Functions were assessed by acidogenicity, adherence strength and sensitivities to anticaries agents. RESULTS: Although both biofilms exhibited a Streptococcus predominant bacterial composition, there were differences in bacterial and EPS compositions; in particular, little glucan EPS was observed in sucrose-independent biofilm. Compared with sucrose-dependent biofilm, acidogenicity, adherence strength and antimicrobial resistance of sucrose-independent biofilm were only slightly lower. However, dextranase degradation was substantially lower in sucrose-independent biofilm. CONCLUSION: Our findings suggest that sucrose-independent biofilm may have cariogenicity as with sucrose-dependent biofilm. These in vitro models can help further elucidate plaque-induced caries aetiology and develop new anticaries agents.

Decreased expression of E-cadherin by Porphyromonas gingivalis-lipopolysaccharide attenuates epithelial barrier function.

BACKGROUND AND OBJECTIVE: The gingival epithelium is a first line of defense against bacterial challenge. E-cadherin (E-cad) plays an important role in cell-cell adhesion as a barrier in the epithelium. Recently, a decrease in the expression of E-cad has been observed in inflamed gingival tissue. The aims of this study were to clarify the changes in E-cad expression and barrier function in human gingival epithelial cells stimulated with Porphyromonas gingivalis-lipopolysaccharide (P. gingivalis-LPS) and to evaluate the influence of these changes on the inflammatory reaction. Furthermore, to clarify the mechanism of the E-cad changes induced by P. gingivalis-LPS, we focused on reactive oxygen species (ROS) that are reported to induce a decrease in E-cad expression. MATERIAL AND METHODS: Human gingival epithelial cells were incubated in Humedia-KG2 in the presence or absence of P. gingivalis-LPS and antioxidants to analyze ROS involvement in P. gingivalis-LPS-induced E-cad changes. E-cad protein expression was analyzed by immunofluorescence staining. To investigate barrier function and inflammatory changes, we performed transport and cytokine assays using gingival epithelial cells and macrophages co-culture model in transwell plates. Medium containing 10 µg/mL P. gingivalis-LPS (transport substance) was added to the upper compartment, which harvested gingival epithelial cells, and medium without P. gingivalis-LPS was added to the lower compartment, which harvested macrophages. In the transport assay, P. gingivalis-LPS penetration was analyzed using the Limulus amebocyte lysate test. In the cytokine assay, we examined the change in tumor necrosis factor-α (TNF-α) production from the macrophages in the lower compartment using enzyme-linked immunosorbent assay. RESULTS: Expression of E-cad in human gingival epithelial cells was decreased by P. gingivalis-LPS, and the decrease in E-cad accelerated the penetration of P. gingivalis-LPS through the monolayer. In addition, the concentration of TNF-α was higher under the E-cad reduced monolayer. Antioxidants, particularly vitamin E and l-ascorbic acid 2-phosphate magnesium salt, inhibited the decrease in E-cad expression, penetration of P. gingivalis-LPS and increase in TNF-α. CONCLUSION: These results suggest that the decrease in E-cad caused by P. gingivalis-LPS leads to destruction of the epithelial barrier function in human gingival epithelial cells, and finally accelerates the inflammatory reaction under the barrier. Antioxidants, particularly vitamin E and l-ascorbic acid 2-phosphate magnesium salt, may restore the impaired function by scavenging ROS, which are related to the decrease in E-cad expression by P. gingivalis-LPS.

Brn-3b inhibits generation of amacrine cells by binding to and negatively regulating DLX1/2 in developing retina.

During retinogenesis, the basic helix-loop-helix proneural gene math5 (atoh7) initiates the generation of the first-born neurons, retinal ganglion cells (RGCs), by activating a network of RGC transcription factors, including Brn-3b (POU4F2). Herein, we show that the expression of DLX1 and DLX2 is significantly down-regulated in math5-null retina but is markedly increased in Brn-3b-null retina. Interestingly, Brn-3b interacts with DLX1 through its homeodomain, and this interaction represses DLX1 activity. Retrovirus-mediated mis-expression of DLX1 or DLX2 dramatically increases the number of amacrine/bipolar cells and concurrently reduces rod photoreceptors. Conversely, combined ectopic expression of Brn-3b with DLX1 or DLX2 promotes the production of RGCs and inhibits amacrine cell differentiation. Thus, DLX1/2 play an essential role in cell fate selection between amacrine and RGCs. Brn-3b suppresses the role of DLX1/2 through physical interaction and biases the competent precursors toward RGC fates.

Evidence for Alfvén waves in solar x-ray jets.

Coronal magnetic fields are dynamic, and field lines may misalign, reassemble, and release energy by means of magnetic reconnection. Giant releases may generate solar flares and coronal mass ejections and, on a smaller scale, produce x-ray jets. Hinode observations of polar coronal holes reveal that x-ray jets have two distinct velocities: one near the Alfvén speed ( approximately 800 kilometers per second) and another near the sound speed (200 kilometers per second). Many more jets were seen than have been reported previously; we detected an average of 10 events per hour up to these speeds, whereas previous observations documented only a handful per day with lower average speeds of 200 kilometers per second. The x-ray jets are about 2 x 10(3) to 2 x 10(4) kilometers wide and 1 x 10(5) kilometers long and last from 100 to 2500 seconds. The large number of events, coupled with the high velocities of the apparent outflows, indicates that the jets may contribute to the high-speed solar wind.

Effect of interferon on incidence of hepatocellular carcinoma in patients with chronic hepatitis C.

BACKGROUND/AIMS: The aim of this study was to evaluate whether IFN prevents the development of HCC in patients with chronic hepatitis C. METHODOLOGY: 103 patients with chronic hepatitis C received IFN and 30 control patients were enrolled in this study. RESULTS: In 33 patients (32.0%) who received IFN, alanine aminotransferase (ALT) decreased to normal range and HCV RNA became negative (complete response: CR). In 7 patients (6.7%), ALT decreased to less than 50 IU/L or stayed within the normal range, but HCV RNA remained positive (biochemical response: BR). In 63 patients (61.1%) and 30 control patients, ALT did not change and HCV RNA remained positive (no response: NR). HCC developed in 5 (4.9%) of the 103 patients who received IFN and 7 (23.3%) of the control patients (p<0.01). In 5 patients who developed HCC, the response to IFN was NR and no HCC developed in patients with CR or BR. In addition, 5-year cumulative rate of development of HCC in 63 IFN NR patients and in control was 7.9% and 23.3% (p<0.05). CONCLUSIONS: IFN decreased the development of HCC in not only patients with CR or BR but also patients with NR.

Infected Charcot spine.

STUDY DESIGN: Case report of an infected Charcot spine following spinal cord injury. OBJECTIVE: To describe this very rare pathological condition and the results of surgical treatment. SETTING: A department of orthopaedic surgery in Japan. METHODS: A 44-year-old man presented with a destructive lesion in the lumbo-sacral spine and a fistula in his back. Anterior bone graft, percutaneous external spinal fixation, and suction/irrigation of the wound were performed. After 4 months, posterior spinal instrumentation surgery was carried out. RESULTS: Primary closure of the fistula and complete bone fusion was achieved after the operation. CONCLUSION: Infection of a Charcot spine, although a rare clinical entity, should be considered as a diagnostic possibility in the spinal cord-injured patients. External spinal fixation is a useful method for the unstable spinal lesion with infection.

Colonic carcinoma resembling submucosal tumor: report of a case and review of the literature.

Submucosal tumor-like colorectal carcinoma, most of whose surface is covered with normal mucosa, is very rare. We report a case of colonic carcinoma resembling submucosal tumor. A 54-yr-old man visited our institution for an evaluation of a positive fecal occult blood test. Colonoscopic examination revealed a small, mainly red polypoid lesion with a central deep ulceration and many white spots in the sigmoid colon. Indigocarmine staining demonstrated that the white spots were faint shallow depressions. Magnifying colonoscopic examination showed that the lesion surface, except for the ulceration and the depressions, was covered with normal mucosa. Although the tumor was small, we strongly suspected its malignancy due to a deep ulceration. As we could not excise it endoscopically, we performed sigmoidectomy. The lesion was 12 mm in size. Histologic examination revealed that the lesion was a moderately differentiated adenocarcinoma that was mainly covered with normal mucosa, that carcinoma was exposed only at the ulceration and the depressions on the surface, and that it had expanded to the muscularis propria. Together with considerations from the literature, this type of colorectal carcinoma is supposed to be invasive and surgical resection should be considered, no matter how small it may be.

A gastric hyperplastic polyp observed endoscopically before and after autoamputation.

Autoamputation of a gastric polyp is a relatively rare phenomenon and its precise mechanism is unclear. To learn more about the mechanism(s) involved, it is important to observe a polyp just before and just after its disappearance. We report a case of a gastric polyp that was observed endoscopically just before and then just after autoamputation. A 61-year-old woman with a thumb-sized, pedunculated hyperplastic polyp in the gastric antrum visited our institution for investigation of hematemesis. She was being treated with oral hypoglycaemic drugs for diabetes mellitus but was not taking any other medicine around that time. Emergency gastroscopy revealed a bleeding point near the polyp; gastroscopy the next day revealed that the polyp had disappeared. It was concluded that autoamputation of a gastric polyp may follow gastric injury induced by diabetes mellitus or oral antidiabetic drugs.

Learning behaviour and cerebral protein kinase C, antioxidant status, lipid composition in senescence-accelerated mouse: influence of a phosphatidylcholine-vitamin B12 diet.

Our objective was to determine whether dietary supplementation with phosphatidylcholine (PC) plus vitamin B12 could afford beneficial effects on biochemical and biophysical events in the brain of senescence-accelerated mouse (SAM) substrain SAMP8. We measured learning behaviour, hippocampal protein kinase C (PKC) activity, cerebral antioxidant status, phospholipid composition and fatty acid composition in 6-month-old SAMP8 and in age-matched controls (SAM substrain SAMR1). In comparison with SAMR1, SAMP8 showed a significant elevation in total grading score of senescence and a significant decline in acquisition SAMP8 had a lower hippocampal PKC activity and cerebral PKC-beta mRNA abundance than SAMR1. SAMP8 had increased cerebral lipid peroxide levels and proportion of sphingomyelin, and a lower proportion of 20 : 4n-6 and 22 : 6n-3 in cerebral phosphtidylethanolamine than SAMR1. SAMP8 fed the PC combined with vitamin B12 diet had an increased PKC activity and a higher proportion of 22 : 6n-3 than SAMP8 fed the control diet. These results indicate the potential benefit of PC combined with vitamin B12 as a dietary supplement.

Comparison of the activity of four chitosan derivatives in reducing initial adherence of oral bacteria onto tooth surfaces.

We examined the effects of four kinds of chitosan derivatives on initial adherence of oral bacteria onto human anterior teeth surfaces. The buccal surfaces of anterior teeth were used as the experimental surfaces. They were divided into five rectangle areas with outer dimensions of about 2 mm x 4 mm. After applying two ml of a sample solution onto the tooth surfaces, an examiner wiped each rectangle area with a sterilized plastic swab one, three and six hours later. Then we measured bacterial counts in sterilized swabs with mitis salivarius agar. We found that the order of magnitude of the inhibitory effect on the adherence of oral bacteria was low molecular chitosan > phosphorylated chitosan > amorphous chitosan > carboxymethyl chitosan. The solution containing 0.5% low molecular chitosan depressed the bacterial adherence to the same extent as a 50 ppm chlorhexidine digluconate solution for three hours, and 0.1% phosphorylated chitosan also exhibited an inhibitory effect in bacterial adherence for one hour. Amorphous chitosan had a moderate inhibitory effect, but no clear inhibitory activity was found with 0.1% carboxymethyl chitosan. These results suggest that low molecular chitosan and phosphorylated chitosan have the potential to effectively inhibit the initial adherence of oral bacteria onto human tooth surfaces.

Effect of rinsing with phosphorylated chitosan on four-day plaque regrowth.

This clinical investigation examined the effect of phosphorylated chitosan rinsing on plaque development and on the buffering capacity of plaque suspension. Three male adult subjects participated in the trial that was designed as a single blind study. Participants refrained from mechanical oral hygiene procedures during a four-day study and rinsed three times a day with 20 ml of test solutions. A wash-out period of three days was instituted between the placebo and phosphorylated chitosan rinsing period. Clinical evaluation and plaque sampling were performed at the end of each test period. We disclosed plaque accumulations on the buccal upper front teeth with a two-tone disclosing agent to distinguish between newly formed plaque and old plaque. After taking color slides, we then used a computerized image analysis. Tooth areas covered by plaque on the color slides were digitized and expressed as percentages of the tooth area. The buffering capacity of the collected plaque fluid was determined by using a beta-titrator. A mouth rinse containing 0.5% phosphorylated chitosan significantly reduced both newly formed plaque areas (red disclosed; p < 0.001) and old plaque areas (blue disclosed; p < 0.01) compared to a placebo rinsing. However there was no significant difference in the plaque buffering capacity (p > 0.05) between the mouth rinse containing 0.5% phosphorylated chitosan and placebo. These findings might suggest that mouth rinse containing phosphorylated chitosan would be effective in reducing plaque formation and have a slight ability to enhance plaque buffering capacity.

A case of superior oblique myokymia observed by an image-analysis system.

Superior oblique myokymia is a microtremor of the eye that causes monocular torsional oscillopsia. A modified Harada-Ito procedure was used to treat a case of the disease in a 20-year-old woman. The authors used video-image analysis pre- and postoperatively to evaluate the effect of the surgery on abnormal torsional eye movements. This analysis revealed that before surgery, the abnormal torsional movement had a very regular cycle (duration of attack, 8.0 +/- 0.5 s; time interval between attacks, 18.7 +/- 3.2 s; n = 9). After the surgery, amplitude of the abnormal torsional eye movement was reduced, and the oscillopsia had subjectively improved, although the movement cycle remained unchanged. The authors' video-image analysis, which used iris striation, proved to be a useful method for clinical measurement of torsional eye movements.

A-type K+ current mediated by the Kv4 channel regulates the generation of action potential in developing cerebellar granule cells.

During neuronal differentiation and maturation, electrical excitability is essential for proper gene expression and the formation of synapses. The expression of ion channels is crucial for this process; in particular, voltage-gated K(+) channels function as the key determinants of membrane excitability. Previously, we reported that the A-type K(+) current (I(A)) and Kv4.2 K(+) channel subunit expression increased in cultured cerebellar granule cells with time. To examine the correlation between ion currents and the action potential, in the present study, we measured developmental changes of action potentials in cultured granule cells using the whole-cell patch-clamp method. In addition to an observed increment of I(A), we found that the Na(+) current also increased during development. The increase in both currents was accompanied by a change in the membrane excitability from the nonspiking type to the repetitive firing type. Next, to elucidate whether Kv4.2 is responsible for the I(A) and to assess the effect of Kv4 subunits on action potential waveform, we transfected a cDNA encoding a dominant-negative mutant Kv4.2 (Kv4.2dn) into cultured cells. Expression of Kv4.2dn resulted in the elimination of I(A) in the granule cells. This result demonstrates that members of the Kv4 subfamily are responsible for the I(A) in developing granule cells. Moreover, elimination of I(A) resulted in shortening of latency before the first spike generation. In contrast, expression of wild-type Kv4.2 resulted in a delay in latency. This indicates that appearance of I(A) is critically required for suppression of the excitability of granule cells during their maturation.

Development of a new device for recording condylar head movement.

Several excellent devices have recently been developed to precisely record mandibular movement. However, these devices are not always suitable for use under certain conditions, such as during sleep, because they incorporate a face bow unit. We report here a newly developed, easy and convenient recording device that does not require the use of an instrument within the mouth. Instead, a subminiature pressure transducer is inserted between the outer surface of a fixation device in the external auditory meatus and the anterior cutaneous surface of the external auditory meatus. The fixation device was made using silicone impression material in the shape of the inner external auditory meatus. Following moulding, the material was cut in half and the parts were reconnected using a coiled spring. This method is based on a routine clinical method for palpating the external auditory meatus to observe condylar head movement. By comparing the results obtained using this device with those obtained using CADIAX, we confirmed that it is useful for recording the movement of the condylar head in routine clinical examinations.

Ibotenic acid-induced lesions of the medial septum increase hippocampal membrane associated protein kinase c activity and reduce acetylcholine synthesis: prevention by a phosphatidylcholine/vitamin B12 diet.

Ibotenic acid infusion into the medial septum (MS) results in biochemical alterations in the hippocampus. The biochemical events involved in this neuronal lesion are poorly understood. We investigated the effect of a purified diet supplemented with egg phosphatidylcholine (PC) and vitamin B(12) on ibotenic acid-medicated biochemical changes in the rat hippocampus and crude synaptosomal membranes. Male Wistar rats with this MS lesion were fed a purified diet (control diet) or a purified diet supplemented with 5.7 g PC and 125 microg vitamin B(12) per 100 g (experimental diet) for 18 days. Sham-operated rats were fed the control diet. Compared with the sham-operated rats, MS-lesioned rats fed the control diet showed increased activity of membrane-bound protein kinase C (PKC), decreased activity of choline acetyltransferase, and decreased concentrations of acetylcholine in the hippocampus. The ratio of cholesterol to phospholipid in the crude synaptic membrane was lower in the lesioned rats than in the sham-operated rats, but this was not accompanied by any alteration in membrane lipid fluidity. MS-lesioned rats fed the experimental diet showed lowered PKC activity and elevated acetylcholine concentrations than did rats fed the control diet, but there were no significant effects on choline acetyltransferase activity and the lipid ratio. The ibotenic acid-mediated elevation of PKC activity was observed as early as 2 days postinjury in the control diet-fed rats but not in the experimental diet-fed rats. We propose that ibotenic acid mediates pathophysiologic actions through the activation of PKC and that PC combined with vitamin B(12) ameliorates the second messenger-mediated injury.

Inhibition of experimental bone resorption and osteoclast formation and survival by 2-aminoethanesulphonic acid.

It is known that bone resorption is mediated by osteoclasts, and lipopolysaccharide (LPS) and inflammatory mediators such as interleukin-1 (IL-1) and prostaglandin E2 (PGE2) induce osteoclast differentiation from haemopoietic cells, 2-aminoethanesulphonic acid, which is known as taurine, is an important nutrient and is added to most synthetic human infant milk formulas. In this study, it was found that 2-aminoethanesulphonic acid inhibits the stimulation of bone resorption mediated by LPS of the periodontopathic microorganism Actinobacillus actinomycetemcomitans Y4 in organ cultures of newborn mouse calvaria. The effect of 2-aminoethanesulphonic acid on the development and survival of osteoclast-like multinucleated cells produced in a mouse bone-marrow culture system was also examined. 2-aminoethanesulphonic acid (100 microg/ml) suppressed the formation of these osteoclast-like cells in the presence of LPS of A. actinomycetemcomitans Y4, IL-1alpha or PGE2 in mouse marrow cultures. On the other hand, 2-aminoethanesulphonic acid did not inhibit 1alpha, 25-dihydroxyvitamin D3-mediated osteoclast differentiation. Although IL-1alpha elongated the survival of the osteoclast-like cells, 2-aminoethanesulphonic acid blocked the supportive effect of IL-1alpha on osteoclast survival. 2-aminoethanesulphonic acid showed no effect on the growth of mouse osteoblasts. Finally, it was found that 2-aminoethanesulphonic acid inhibited alveolar bone resorption in experimental periodontitis in hamsters. These results suggest that 2-aminoethanesulphonic acid is an effective agent in preventing inflammatory bone resorption in periodontal diseases.

Primary duodenal MALT lymphoma.

Primary duodenal MALT lymphoma (MALToma) is a very rare neoplasm arising from the mucosa-associated lymphoid tissue of the duodenum. We report a 55-year-old woman with MALToma located in the descending duodenum and accompanying Helicobacter pylori infection of the stomach. We performed operative resection due to involvement of the papilla of Vater and submucosal tumor infiltration. Despite wide mucosal spreading, postoperative examination revealed only a small amount of MALToma cells infiltrating into the submucosa. No invasion into the adjacent structure or metastasis to regional lymph nodes was confirmed, suggesting the disease could have been controlled by eradication of Helicobacter pylori.

Serum des-gamma-carboxyprothrombin level by a modified enzyme immunoassay method in hepatocellular carcinoma: clinical significance in small hepatocellular carcinoma.

BACKGROUND/AIMS: In the diagnosis of hepatocellular carcinoma (HCC), serum des-gamma-carboxyprothrombin (DCP) is a useful tumor marker. The conventional immunoassays for measurement of DCP levels, however, are not sensitive enough to detect small HCC. Therefore, we intended to elevate the minimal detection level of DCP by a modified enzyme linked immunosorbent assay method. METHODOLOGY: This modified assay method is similar to the conventional enzyme linked immunosorbent assay (ELISA) method, but the first reaction occurs overnight. As a result, the minimal detection levels of DCP varied from 0.06 AU/ml, by the conventional method, to 0.008 AU/ml, by the modified method. Two hundred and twenty five serum samples from 100 patients with HCC, 75 with liver cirrhosis and 50 with chronic hepatitis were subjected to the present study. Simultaneous determinations of serum DCP by the modified assay and a-fetoprotein (AFP) levels were performed. RESULTS: Eighty five of 100 patients with HCC had increased DCP levels of more than 0.008 AU/ml. This method yielded a sensitivity of 85%, a specificity of 90% and a total accuracy value of 88%. In 27 patients with small HCC (less than 30 mm in diameter), 12 had elevated DCP levels, resulting in a sensitivity of 44%. When the modified DCP assay together with AFP measurement (more than 20 ng/ml) were introduced, the sensitivity was 67% in the 27 patients with small HCC. CONCLUSIONS: This modified ELISA method increased the sensitivity in patients with small HCC, and the combination assay of serum DCP and AFP levels was more useful for the early diagnosis of HCC.

[Drug susceptibility of clinically isolated Helicobacter pylori].

Between January 1995 and March 1997, 78 Helicobacter pylori strains were isolated from patients with gastritis and gastric ulcer and their drug-susceptibilities to 8 antimicrobial agents and 3 anti-ulcer drugs were determined. Imipenem was the most active agent and its MICs to all the strains tested were lower than 0.013 microgram/ml. Amoxicillin, cefaclor and minocycline were active against H. pylori with MIC90s of 0.05 microgram/ml, 0.78 microgram/ml and 0.39 microgram/ml, respectively, and no resistant strains against these drugs were isolated. However, resistant strains to clarithromycin (isolation frequency: 9%), erythromycin (13%), ofloxacin (8%) and metronidazole (13%) were isolated. Triple, double and single resistant strains to above 4 antimicrobial agents were noted. No quadruple resistant strain was isolated. Frequencies of those resistance patterns were 14.3% (triple), 28.6% (double), and 57.1% (single), respectively. Seven erythromycin-resistant strains were shown to be cross-resistant to clarithromycin but 3 erythromycin-resistant strains were susceptible to clarithromycin. It seems likely that this phenomenon is caused by the fact that clarithromycin is more active to H. pylori than erythromycin. The MIC90 value of lansoprazole was lower than those of omeprazole and famotidine.