Characterization of a Treponema denticola ATCC 35405 mutant strain with mutation accumulation, including a lack of phage-derived genes.

Trepoenema denticola, a spirochetal bacterium, is associated with periodontal diseases. The type strain of the bacterium, ATCC 35405, is commonly used in a basic research. Here, we report that our stock strain derived from ATCC 35405 had a mutation on the chromosome and expressed differential characteristics from the original strain. Genome sequencing analysis revealed the lack of a phage-derived region, and over 200 mutations in the mutant strain. The mutant grew to a higher density in broth culture as compared with the origin. In addition, the mutant formed a colony on the surface of the agar medium, whereas the origin could not. On contrary, the mutant showed decreased motility and adhesion to gingival epithelial cells. There were no differences in the bacterial cell length and a chymotrypsin-like protease activity between the two strains. RNA and genome sequencing analysis could not identify the genes that introduced the phenotypic differences between the strains. This mutant is potentially useful for examining the genetic background responsible for the physiological and pathogenic characteristics of T. denticola.

Draft Genome Sequence of Neisseria mucosa Strain HSUH001, Isolated from an Aggressive Periodontal Lesion.

We report the draft genome sequence (143 contigs, with a total length of 2,424,805 bp and an N 50 value of 36,066 bp) of a bacterium isolated from an aggressive periodontal lesion in a patient. We assigned strain HSUH001 to Neisseria mucosa through a multilocus sequence analysis.

Fructo-oligosaccharides ameliorate steatohepatitis, visceral adiposity, and associated chronic inflammation via increased production of short-chain fatty acids in a mouse model of non-alcoholic steatohepatitis.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a hepatic manifestation of metabolic syndrome. Within the spectrum of NAFLD, non-alcoholic steatohepatitis (NASH) in combination with hepatic inflammation and fibrosis can lead to liver cirrhosis and hepatocellular carcinoma. Dysbiosis was reported to contribute to NASH pathogenesis. This study aimed to determine the effects of fructo-oligosaccharides (FOS) on steatohepatitis and visceral adiposity in an obese mouse model of NASH. METHODS: Twelve newborn C57BL/6 J male mice were subcutaneously injected with monosodium glutamate (MSG) to induce obesity on a conventional diet. Six mice were also administered 5% FOS via drinking water from 10 weeks of age. At 18 weeks, histological characteristics of the liver and epididymal fat were compared between the groups. Hepatic mRNA expression of lipid metabolism enzymes and SCFA in feces and sera were measured. RESULTS: Hepatic steatosis, inflammatory cell infiltration, and hepatocyte ballooning in the liver and increased hepatic mRNA expression of fatty acid synthase and glycerol-3-phosphate acyltransferase were observed in the MSG-treated mice. FOS treatment improved the liver pathology and blunted the increases in the mRNA expression levels of lipid metabolism enzymes. In addition, FOS inhibited adipocyte enlargement and formation of crown-like structures and reduced the M1 macrophage frequency in the epididymal fat of the MSG mice (39.4% ± 3.0% vs. 22.8% ± 0.7%; P = 0.001). FOS increased not only the fecal concentrations of n-butyric acid (0.04 ± 0.01 vs. 0.38 ± 0.14 mg/g, P = 0.02), propionic acid (0.09 ± 0.03 vs. 0.42 ± 0.16 mg/g, P = 0.02), and acetic acid (0.65 ± 0.16 vs. 1.48 ± 0.29 mg/g, P = 0.03) but also the serum concentration of propionic acid (3.9 ± 0.5 vs. 8.2 ± 0.5 µmol/L, P = 0.001). CONCLUSIONS: FOS ameliorates steatohepatitis, visceral adiposity, and chronic inflammation by increasing SCFA production.

Immunoglobulin G4-related Liver Disease Overlapping with Non-alcoholic Steatohepatitis That Was Diagnosed Simultaneously with Autoimmune Pancreatitis: A Case Report and Review of the Literature.

A 70-year-old woman was referred to our hospital due to symptoms of dry eyes, dry mouth, and epigastric pain. Computed tomography showed distal pancreatic swelling, liver edge dullness and surface irregularities. Serum anti-nuclear antibody titers, immunoglobulin G and IgG4 levels were elevated. Autoimmune pancreatitis (AIP) was diagnosed based on endoscopic findings and a histopathological examination. Her AIP improved after starting prednisolone treatment. A liver biopsy revealed interface hepatitis with lymphoplasmacyte and IgG4-positive plasma cell infiltration. In addition, non-alcoholic steatohepatitis (NASH) was diagnosed based on the presence of parenchymal steatosis, ballooning hepatocytes, and pericellular fibrosis. We experienced a unique liver disease case showing IgG4-related liver disease overlapping with NASH.

Detection of Anti-mitochondrial Antibodies Accompanied by Drug-induced Hepatic Injury due to Atorvastatin.

A 44-year-old Japanese woman was admitted to our hospital with fatigue and an altered liver function. She had been receiving atorvastatin treatment for 10 months. Although no jaundice was seen, the patient's serum alkaline phosphatase and γ-glutamyl transpeptidase levels were markedly elevated. Based on the results of a drug-induced lymphocyte-stimulation test, her liver disease was diagnosed as atorvastatin-induced hepatic injury. Subsequently, anti-mitochondrial antibodies (AMAs) were detected in her serum; however, a liver biopsy specimen did not show the characteristic features of primary biliary cholangitis. We herein report the detection of AMAs accompanied by drug-induced hepatic injury caused by atorvastatin.

[Successful Hepatic Arterial Injection Chemotherapy for Hepatocellular Carcinoma with Inferior Vena Cava Invasion and Extrahepatic Spread in a Patient with Child-Pugh Class BLiver Cirrhosis].

A 65-year-old woman who had liver cirrhosis(Child-Pugh class B)due to hepatitis C infection was diagnosed with hepatocellular carcinoma with hepatic vein invasion, portal vein tumor invasion, and lung metastasis. No recommended treatment was noted in the clinical practice guidelines for hepatocellular carcinoma with vascular invasion in patients with Child- Pugh class B liver cirrhosis. After initiating arterial injection chemotherapy, marked decreases in tumor size of lung metastasis, vascular invasion, and primary liver cancer were observed. Based on our experience and previous reports, hepatic arterial infusion chemotherapy was considered valuable for hepatocellular carcinoma with vascular invasion, even in patients with Child-Pugh class B liver cirrhosis.

Identification of an early stage biofilm inhibitor from Veillonella tobetsuensis.

Oral biofilm, the cause of dental caries and periodontal diseases, consists of multiple bacterial species. Streptococcus spp. and Veillonella spp. have been reported as to be initial and early colonizers of oral biofilms. Our previous studies showed that Veillonella tobetsuensis may play an important role on the development of S. gordonii biofilms without coaggregation involving extracellular biomolecules. In this study, the effect of a cyclic dipeptide autoinducer from culture supernatants from V. tobetsuensis at late-exponential growth phase on S. gordonii biofilm was examined. The cyclic dipeptide, identified as cyclo (-L-Leu-L-Pro) by gas chromatography/mass spectrometry, inhibited the development of S. gordonii biofilm. Furthermore, cyclo (-L-Leu-L-Pro) appeared not to cause bactericidal effects on planktonic cells of S. gordonii. This is the first report that oral Veillonella produces cyclo (-L-Leu-L-Pro) in their culture supernatants. Moreover, the results of this study suggest that cyclo (-L-Leu-L-Pro) may have an application to inhibit early stage development of oral biofilms.

Development of Autoimmune Hepatitis during Direct-acting Antiviral Therapy for Chronic Hepatitis C Virus Infection.

An 81-year-old woman developed liver dysfunction after two months' treatment with direct-acting antivirals (DAAs) for chronic hepatitis C virus (HCV) infection. She was positive for serum anti-nuclear antibody, with an elevated immunoglobulin G level. A liver biopsy revealed high-grade interface hepatitis and infiltrate of lymphocytes and plasma cells. DAA-associated drug-induced autoimmune hepatitis (DI-AIH) was considered. Her liver dysfunction improved after discontinuing DAA therapy and starting prednisolone treatment. The differential diagnosis for AIH should include liver injury during DAA therapy for chronic HCV infection.

Veillonella infantium sp. nov., an anaerobic, Gram-stain-negative coccus isolated from tongue biofilm of a Thai child.

A strain of a novel anaerobic, Gram-stain-negative coccus was isolated from the tongue biofilm of a Thai child. This strain was shown, at the phenotypic level and based on 16S rRNA gene sequencing, to be a member of the genus Veillonella. Comparative analysis of the 16S rRNA, dnaK and rpoB gene sequences indicated that phylogenetically the strain comprised a distinct novel branch within the genus Veillonella. The novel strain showed 99.8, 95.1 and 95.9 % similarity to partial 16S rRNA, dnaK and rpoB gene sequences, respectively, to the type strains of the two most closely related species, Veillonelladispar ATCC 17748T and Veillonellatobetsuensis ATCC BAA-2400T. The novel strain could be discriminated from previously reported species of the genus Veillonella based on partial dnaK and rpoB gene sequencing and average nucleotide identity values. The major acid end-product produced by this strain was acetic acid under anaerobic conditions in trypticase-yeast extract-haemin with 1 % (w/v) glucose or fructose medium. Lactate was fermented to acetic acid and propionic acid. Based on these observations, this strain represents a novel species, for which the name Veillonella infantium sp. nov. is proposed. The type strain is T11011-4T (=JCM 31738T=TSD-88T).

Fructo-oligosaccharides and intestinal barrier function in a methionine-choline-deficient mouse model of nonalcoholic steatohepatitis.

Impairments in intestinal barrier function, epithelial mucins, and tight junction proteins have been reported to be associated with nonalcoholic steatohepatitis. Prebiotic fructo-oligosaccharides restore balance in the gastrointestinal microbiome. This study was conducted to determine the effects of dietary fructo-oligosaccharides on intestinal barrier function and steatohepatitis in methionine-choline-deficient mice. Three groups of 12-week-old male C57BL/6J mice were studied for 3 weeks; specifically, mice were fed a methionine-choline-deficient diet, a methionine-choline-deficient diet plus 5% fructo-oligosaccharides in water, or a normal control diet. Fecal bacteria, short-chain fatty acids, and immunoglobulin A (IgA) levels were investigated. Histological and immunohistochemical examinations were performed using mice livers for CD14 and Toll-like receptor-4 (TLR4) expression and intestinal tissue samples for IgA and zonula occludens-1 expression in epithelial tight junctions. The methionine-choline-deficient mice administered 5% fructo-oligosaccharides maintained a normal gastrointestinal microbiome, whereas methionine-choline-deficient mice without prebiotic supplementation displayed increases in Clostridium cluster XI and subcluster XIVa populations and a reduction in Lactobacillales spp. counts. Methionine-choline-deficient mice given 5% fructo-oligosaccharides exhibited significantly decreased hepatic steatosis (p = 0.003), decreased liver inflammation (p = 0.005), a decreased proportion of CD14-positive Kupffer cells (p = 0.01), decreased expression of TLR4 (p = 0.04), and increases in fecal short-chain fatty acid and IgA concentrations (p < 0.04) compared with the findings in methionine-choline-deficient mice that were not administered this prebiotic. This study illustrated that in the methionine-choline-deficient mouse model, dietary fructo-oligosaccharides can restore normal gastrointestinal microflora and normal intestinal epithelial barrier function, and decrease steatohepatitis. The findings support the role of prebiotics, such as fructo-oligosaccharides, in maintaining a normal gastrointestinal microbiome; they also support the need for further studies on preventing or treating nonalcoholic steatohepatitis using dietary fructo-oligosaccharides.

Henoch Schönlein Purpura Nephritis Associated with Intravesical Bacillus Calmette-Guerin (BCG) Therapy.

Henoch Schönlein purpura (HSP), also known as IgA vasculitis (IgAV), is a systemic small-vessel vasculitis that predominantly affects adolescents and is rare in adults. In many cases, the onset of HSP has been causally linked to an infectious disease. We encountered a case of HSP with severe renal involvement diagnosed by renal biopsy following bacillus Calmette-Guerin (BCG) therapy for bladder cancer. This is of clinical relevance, as intravesical BCG administration is becoming an established therapy for superficial bladder cancer and is supposed to be safe. It is important for all clinicians to recognize that BCG therapy has this rare but potentially serious systemic complication.

Effects of chitosan fiber addition on the properties of polyurethane with thermo-responsive shape memory.

We investigated the effects of the addition of chitosan fiber (biomass nanofiber made by Sugino (BiNFi-s)) to polyether-based thermoplastic polyurethane (TPU) on material properties. BiNFi-s (2 and 5 wt %)/TPU composite materials were prepared via compression molding, and glass fiber (2 and 5 wt %)/TPU composite materials and plain TPU were also prepared for comparison. The glass transition temperature was analyzed using differential scanning calorimetry, and the crystal structure was investigated using X-ray diffraction. 20-mm-long test specimens with cross-sectional dimensions of 1 mm × 1 mm were cut from sheets of the composite materials, and three-point bending tests were carried out using a universal testing machine to investigate their mechanical properties and shape memory. The addition of BiNFi-s or glass fiber to TPU did not influence the glass transition temperature, although the crystal structure changed from semi-crystalline to amorphous. The elastic modulus increased 40% by the addition of 5 wt % BiNFi-s (2.31 MPa) compared with plain TPU (1.65 MPa), and these composites exhibited shape recovery with clinically relevant changes in temperature. The addition of 5 wt % BiNFi-s into TPU resulted in an improvement in the elastic modulus without any decrease in the shape memory effect. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 1151-1156, 2017.

Efficacy of nalfurafine hydrochloride in patients with chronic liver disease with refractory pruritus: A randomized, double-blind trial.

AIMS: Patients with chronic liver disease sometimes develop cholestasis, which induces severe whole-body pruritus that may disrupt daily activities and sleep. To determine the efficacy of nalfurafine hydrochloride (5 µg), which is a selective κ-opioid receptor agonist, in improving pruritus, we undertook a double-blind placebo-controlled study in patients with chronic liver disease with refractory pruritus. Nalfurafine hydrochloride at 2.5 µg was also used to evaluate the dose-response relationship. METHODS: In total, 318 subjects were randomly assigned to receive the placebo or nalfurafine hydrochloride (2.5 or 5 µg) given orally once daily for 84 consecutive days. Pruritus was assessed based on the visual analog scale and pruritus scores. RESULTS: Changes in the visual analog scale at week 4 (last observation carried forward) were significantly greater in the nalfurafine hydrochloride groups at 28.56 and 27.46 mm in the 2.5 µg and 5 µg groups, respectively, compared to 19.25 mm in the placebo group (P = 0.0022 and 0.0056, respectively). The major adverse drug reactions (ADRs) included pollakiuria (including nocturia), somnolence, insomnia (including middle insomnia), and constipation. Most ADRs were mild. CONCLUSIONS: Nalfurafine hydrochloride (2.5 or 5 µg daily) was effective in the treatment of refractory pruritus in patients with chronic liver disease. Furthermore, no clinically significant ADRs were observed at either dose.

Effect of roselle calyx extract on in vitro viability and biofilm formation ability of oral pathogenic bacteria.

OBJECTIVE: To investigate the effect of the roselle calyx extract (RCE) (Hibiscus sabdariffa L.) on the in vitro viability and biofilm formation ability of oral pathogenic bacteria. METHODS: RCE was prepared by soaking roselle calyx powder with ethyl alcohol for 24 h at room temperature. After centrifugation, the extract was lyophilized. Then, the extract was dissolved in phosphate-buffered saline, the pH was adjusted, and the extract was aseptically filtered. We used Streptococcus mutans, Streptococcus sanguinis, Lactobacillus casei, Actinomyces naeslundii, Aggregatibacter actinomycetemcomitans, Fusobacterium nucleatum, Porphyromonas gingivalis and Prevotella intermedia in this study. The antibacterial activity of the RCE was determined by treating the cells of these bacteria with the extract for 10 or 20 min at room temperature. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration was determined using the microdilution method, and the effect of the RCE on the ability to form biofilm was determined using a polystyrene micro plate assay. In addition, we used the WST-1 assay to determine the cytotoxicity of the RCE on HGF, Ca9-22 and KB cells. RESULTS: The RCE had antibacterial activity against oral bacteria used in this study. In particular, most significant antibacterial activity was observed against Fusobacterium nucleatum, Prevotella intermedia and Porphyromonas gingivalis. The MIC and minimum bactericidal concentration were 7.2 mg/mL-28.8 mg/mL and 14.4 to >57.6 mg/mL. The RCE had an inhibitory effect on biofilm formation at the MIC and sub-MIC levels. In addition, the RCE had low cytotoxic effects on HGF, Ca9-22 and KB cells. CONCLUSIONS: Thus, our results indicate that the RCE may be used for preventing oral diseases.

Inhibition of initial bacterial adhesion on titanium surfaces by lactoferrin coating.

Because dental implant abutments are located at transmucosal sites, their surface should inhibit bacterial accumulation to prevent peri-implantitis. The authors examined the effects of human lactoferrin (LF), an antibacterial protein present in saliva, as an antibacterial coating on the titanium surface and evaluated its effects before and after mucin-containing artificial saliva (AS) incubation. In the control group, titanium disks were soaked in distilled water, whereas in the LF group, titanium disks were soaked in LF solution to coat the disks. In the control-AS and LF-AS groups, half of the control and LF disks were incubated with AS. To confirm LF adsorption, the fluorescence intensity of fluorescein isothiocyanate-labeled LF was measured. The LF and LF-AS groups showed significantly higher intensity than the control and control-AS groups (P < 0.01). There was no significant difference between the LF and LF-AS groups (P > 0.05). The amount of adhered Streptococcus gordonii significantly increased by incubation with AS (P < 0.01) and significantly decreased by adsorption of LF (P < 0.01). There was no interaction between the two factors, LF adsorption and AS incubation (P = 0.561). These results suggest that the adsorbed LF inhibited bacterial adhesion following AS incubation. According to qualitative LIVE/DEAD analysis, viable bacteria appeared to be decreased in the presence of LF and SEM observation indicated that altered morphologies increased in LF and LF-AS groups. These results suggest that the adsorbed LF remained on the titanium surface after incubation with AS, and the remaining LF inhibited bacterial adhesion and exhibited bactericidal effects. Therefore, the adsorption of LF on the abutment material appears to be effective in preventing peri-implantitis.

[Efficacy and safety of sugammadex (Org 25969) in reversing moderate neuromuscular block induced by rocuronium or vecuronium in Japanese patients].

BACKGROUND: Efficacy and safety of sugammadex in reversing neuromuscular block induced by rocuronium or vecuronium were investgated in Japanese patients. METHODS: We studied 98 Japanese patients undergoing surgery requiring general anesthesia. Patients were allocated randomly to receive intubation dose of rocuronium or vecuronium. During surgery, patients received additional doses of rocuronium or vecuronium for maintenance of moderate block. At T2 reappearance sugammadex 0-4.0 mg . kg-1 was administered. The neuromuscular block was monitored with acceleromyography using TOF stimuli. Sevoflurane was administered to all treatment groups after intubation. RESULTS: For the rocuronium-induced neuromuscular block, the mean recovery time of the T4/T1 ratio to 0.9 decreased from 82.1 min in the placebo group to 1.8 min in the 4.0 mg . kg-1 sugammadex group. For the vecuronium-induced neuromuscular block, it decreased from 83.2 min in the placebo group to 2.1 min in the sugammadex 4.0 mg . kg-1 group. Plasma concentrations of sugammadex were approximately dose proportional over the dose range of 0.5 to 4.0 mg . kg-1 and independent of the neuromuscular blocking agents used. No clinical evidence of recurarization or residual curarization was observed. CONCLUSIONS: The efficacy and safety of sugammadex were confirmed in Japanese surgical patients.

Serological and Histological Examination of a Nonalcoholic Steatohepatitis Mouse Model Created via the Administration of Monosodium Glutamate.

The administration of monosodium glutamate (MSG) to mice induces hepatic steatosis and inflammation. In this study, we investigated the metabolic features of MSG-treated mice and the histological changes that occur in their livers and adipose tissue. MSG mice were prepared by subcutaneously injecting MSG into newborn C57BL/6J male mice. The control mice were subcutaneously injected with saline. Another group of mice was fed a methionine- and choline-deficient diet (MCD). Compared with the control mice, the MSG mice had higher serum levels of insulin and cholesterol than the control mice, whereas the opposite was true for the MCD mice. Microvesicular steatosis and inflammatory cell infiltration were detected in both the MSG and MCD mouse livers. Enlarged adipocytes and crown-like structures were observed in the epididymal fat of the MSG mice, whereas neither of these features was seen in the MCD mice. Flow cytometric analysis revealed increased frequencies of monocytes and M1 macrophages in the livers and epididymal fat tissue of the MSG mice, respectively. The MSG mice exhibited the characteristic liver histopathology of nonalcoholic steatohepatitis (NASH) as well as metabolic syndrome-like features, which suggested that MSG mice are a better model of human NASH than MCD mice.

Lipopolysaccharide extracted from Porphyromonas gingivalis induces DNA hypermethylation of runt-related transcription factor 2 in human periodontal fibroblasts.

BACKGROUND/PURPOSE: Epigenetic alterations such as DNA methylation and histone acetylation are described as changes in the pattern of gene expression not involving the DNA sequence. Lipopolysaccharide (LPS) derived from Porphyromonas gingivalis has been shown to inhibit osteoblastic cell differentiation. We examined whether DNA hypermethylation was involved in the inhibitory effect of LPS on osteoblastic differentiation of fibroblasts derived from human periodontal ligament (HPDL). METHODS: The HPDL cells were incubated with LPS derived from P. gingivalis at a concentration of 10 µg/ml for 24 h. The cells were treated with DNA methyltransferase inhibitor 5-Aza-2'-deoxycytidine (5Aza). Untreated cells were used as a control. Cell viability was determined using cell proliferation reagent. DNA methyltransferase (DNMT1) and runt-related transcription factor 2 (RUNX2) mRNAs were evaluated by quantitative polymerase chain reaction (RT-PCR). Analysis of RUNX2 DNA methylation was performed using quantitative methylation-specific PCR. RESULTS: The expression level of RUNX2 was significantly lower in the cells stimulated with LPS than the controls. The presence of 5Aza increased the expression of RUNX2 in cells stimulated with LPS. The expression levels of DNMT1 mRNA in the cells stimulated with LPS were significantly higher than in the control. The presence of 5Aza completely abolished the upregulated expression of DNMT1 in cells stimulated with LPS. The methylation of DNA at 0.1 kb and -1.9 kb in the cells stimulated with LPS was significantly higher than the control. CONCLUSION: The results indicate that DNA hypermethylation may be involved in the inhibitory effect of LPS on osteoblastic differentiation in HPDL.