Effect of the combined use of enamel matrix derivative and atelocollagen sponge scaffold on osteoblastic differentiation of mouse induced pluripotent stem cells in vitro.

BACKGROUND AND OBJECTIVE: Induced pluripotent stem cells (iPSCs) are a candidate cell source in periodontal regenerative therapy. Enamel matrix derivative (EMD) has been shown to regenerate periodontal tissues, and atelocollagen sponge (ACS) is considered a suitable scaffold or carrier for growth factors. This study aimed to investigate the effect of combined use of EMD and an ACS scaffold on cell behaviors and differentiation of mouse iPSCs (miPSCs) in vitro. MATERIAL AND METHODS: Following embryonic body formation from miPSCs, dissociated cells (miPS-EB-derived cells) were seeded onto ACS with or without EMD, and cultured in osteoblast differentiation medium. Scanning electron microscopy and histological analyses were used to assess cell morphology and infiltration within the ACS. Cell viability (metabolism) was determined using an MTS assay, and expression of mRNA of osteoblastic differentiation markers was assessed by quantitative RT -PCR. Alkaline phosphatase (ALP) staining intensity and activity were evaluated. Mineralization was assessed by von Kossa staining, and calcium content was quantitated using the methylxylenol blue method. RESULTS: By 24 hours after seeding, miPS-EB-derived cells in both the EMD and control groups had attached to and infiltrated the ACS scaffold. Scanning electron microscopy images revealed that by day 14, many cytoplasmic protrusions and extracellular deposits, suggestive of calcified matrix, were present in the EMD group. There was a time-dependent increase in cell viability up to day 3, but no difference between groups was observed at any time point. The levels expressed of ALP and osterix genes were significantly higher in the EMD group than in the control group. Expression of runt-related transcription factor 2 was increased in the EMD group compared with the control group on day 7. EMD upregulated the expression of bone sialoprotein and osteopontin on day 14, whereas expression of osteocalcin was lower at all time points. The staining intensity and activity of ALP were higher in the EMD group than in the control group. Mineralization levels and calcium contents were significantly higher in the EMD group throughout the observation period. CONCLUSION: These data suggest that combining ACS with EMD increases levels of osteoblastic differentiation and mineralization in miPS-EB-derived cells, compared with ACS used alone.

Utility of the FIB-4 Index for hepatocarcinogenesis in hepatitis C virus carriers with normal alanine aminotransferase levels.

The FIB-4 index is a simple formula using age, aspartate aminotransferase, alanine aminotransferase (ALT) and platelet count to evaluate liver fibrosis. We investigated the ability of the FIB-4 index for hepatocarcinogenesis in hepatitis C virus (HCV) carriers with normal ALT levels. A total of 516 patients with ALT levels persistently at or below 40 IU/L during an observation period of over 3 years were included. Factors associated with the development of HCC were determined. Hepatocellular carcinoma (HCC) developed in 60 of 516 patients (11.6%). The incidence rate of HCC at 5 and 10 years was 2.6% and 17.6%, respectively. When patients were categorized according to the FIB-4 index as ≤ 2.0 (n = 226), >2.0 and ≤ 4.0 (n = 169), and > 4.0 (n = 121), the cumulative incidence of HCC at 5 years was 0.5%, 1.3% and 8.0%, respectively, and 2.8%, 25.6% and 37.1% at 10 years, respectively. Patients with FIB-4 index >4.0 were at the highest risk (P < 0.001). Factors that were significantly associated with HCC in the multivariate analysis were FIB-4 index >2.0 (hazard ratio (HR), 7.690), FIB-4 index >4.0 (HR, 8.991), α-fetoprotein (AFP) >5 ng/mL (HR, 2.742), AFP >10 ng/mL (HR, 4.915) and total bilirubin >1.2 mg/dL (HR, 2.142). A scoring system for hepatocarcinogenesis that combines the FIB-4 index and AFP predicted patient outcomes with excellent discriminative ability. The FIB-4 index is strongly associated with the risk of HCC in HCV carriers with normal ALT levels.

Stretching stimulates fibulin-5 expression and controls microfibril bundles in human periodontal ligament cells.

BACKGROUND AND OBJECTIVE: The elastic fiber system comprises oxytalan, elaunin and elastic fibers, differing in their relative microfibril and elastin contents. Human periodontal ligaments contain oxytalan fibers (pure microfibrils). Periodontal ligaments are continuously exposed to various functional forces, such as tooth movement and occlusal loading. We have reported that bundles of microfibrils coalesce in response to mechanical strain in cultured periodontal ligament fibroblasts, as assessed in terms of their positivity for fibrillin-1 (the major component of microfibrils). However, the mechanism of microfibril coalescence is unclear. We hypothesized that the fibrillin-1-binding molecule, fibulin-5, contributes to oxytalan fiber formation under mechanical strain. MATERIAL AND METHODS: We subjected periodontal ligament fibroblasts to stretching in order to examine the effects of fibulin-5 on the formation of oxytalan fibers in cell/matrix layers. We transfected periodontal ligament cells with small interference RNA for fibulin-5, then examined oxytalan fibers using immunofluorescence and electron microscopy. RESULTS: Immunofluorescence showed that fibrillin-1-positive microfibrils coalesced as a result of stretching, compared with cells that were not subjected to stretching. Fibulin-5 colocalized on fibrillin-1-positive microfibrils. Stretching increased fibulin-5 gene expression and protein deposition. Immunofluorescence and immunogold electron microscopy analysis revealed that fibulin-5 suppression inhibited the coalescence of microfibrils under stretching conditions. CONCLUSION: These results suggest that fibulin-5 up-regulated in response to tension strain may control the formation of microfibril bundles in periodontal ligament.

Correlation of serum ribavirin concentration with pretreatment renal function estimates in patients with chronic hepatitis C receiving combination antiviral therapy with peginterferon and ribavirin.

Serum ribavirin concentration is an important factor in antiviral therapy in combination with peginterferon (PEG-IFN) and ribavirin for patients with chronic hepatitis C in terms of both beneficial and adverse effects. We evaluated whether the serum ribavirin concentration can be predicted on the basis of renal function estimates. Serum creatinine and cystatin C concentrations were measured at the start of treatment in a total of 148 patients with chronic hepatitis C who underwent combination PEG-IFN and ribavirin therapy. Creatinine clearance (CrCl) and total clearance of ribavirin (CL/F) were calculated on the basis of the serum creatinine level. The glomerular filtration rate was calculated with two different formulae on the basis of the serum cystatin C level. These values were compared with serum ribavirin concentrations 4 weeks after the start of therapy. The cystatin C level increased with the progression of liver fibrosis, whereas the creatinine level was constant regardless of the degree of liver fibrosis. Significant correlation was not observed between the serum ribavirin concentration and serum creatinine level, cystatin C level, or calculated renal function estimates. However, significant correlation was found between the serum ribavirin concentration and CrCl and CL/F in patients who were given ribavirin >800 mg/day. Overall, renal function estimates do not correlate with the serum ribavirin concentration in Japanese patients with chronic hepatitis C who undergo combination PEG-IFN and ribavirin therapy. Serum creatinine-based renal function estimates might be predictive for the serum ribavirin concentration only in patients with a daily ribavirin intake of 800 mg or more.

A fluorinated 2-nitroimidazole, KU-2285, as a new hypoxic cell radiosensitizer.

To develop new hypoxic cell radiosensitizers, we incorporated fluorine atoms into the side chain of the 2-nitroimidazole. Of the resulting compounds, KU-2285 (a 2-nitroimidazole with an N1-substituent of CH2CF2CONHCH2-CH2OH) was considered the most useful as a hypoxic cell radiosensitizer. In this study, its in vivo radiosensitizing activity and acute toxicity were compared with those of etanidazole. The reduction potentials of KU-2285 and etanidazole were -0.96 V and -1.05 V vs Ag/Ag+ in N,N-dimethylformamide, respectively, and their respective octanol/water partition coefficients were 0.25 and 0.040. The in vivo radiosensitizing activity of KU-2285 was found to be similar to that of etanidazole at the same administration dose when assayed by an in vivo-in vitro assay, a growth delay assay, and a tumor control assay using SCC VII tumor or transplanted mammary tumor in C3H/He mice. Although the radiosensitizing activity of etanidazole was reduced when it was administered orally, there was no significant difference in the radiosensitizing activity of KU-2285 whether it was administered intravenously, intraperitoneally, or orally. The acute toxicity measured as the LD50/7 in 8-week-old female C3H/HeJ mice was found to be 2.4 g/kg (intravenously), 2.1 g/kg (intraperitonealy), and 4.25 g/kg (orally) for KU-2285, whereas it was 4.75 g/kg (intravenously) for etanidazole.

Characteristics of fluorinated nitroazoles as hypoxic cell radiosensitizers.

Types of 2-nitroimidazoles and 3-nitro-1,2,4-triazoles bearing one or two fluorine atoms on their side chains were synthesized to evaluate their physicochemical properties, radiosensitizing effects, and toxicity. The reduction potential of the compounds containing one fluorine was similar to that of misonidazole (MISO), whereas that of the difluorinated compounds was slightly higher. Both mono- and difluorinated compounds had an in vitro sensitizing activity comparable to or slightly higher than that of MISO. The fluorinated 3-nitrotriazoles were almost as efficient as the 2-nitroimidazoles with the same substituent. In vivo, some of the compounds were up to twice more efficient than MISO, whereas others were as efficient as MISO. Toxicity in terms of LD50/7 in mice was quite variable depending on the side-chain structure; the amide derivatives were less toxic than MISO, whereas the alcohol and ether derivatives were more toxic. In view of the radiosensitizing effect and toxicity in vivo, at least one compound, KU-2285 (a 2-nitroimidazole with an N1-substituent of: CH2CF2CONHCH2CH2OH) has been found to be as useful a hypoxic cell sensitizer as SR-2508.

Implication of nitric oxide synthase activity in the genesis of water immersion stress-induced gastric lesions in rats: the protective effects of FK506.

BACKGROUND: Nitric oxide (NO) is a potent cytoprotective substance of gastric mucosa. FK506, an immunosuppressive drug, shows anti-gastric ulcer effects equivalent to famotidine, an H2 blocker, in rats. This study was designed to evaluate the cytoprotective mechanism of FK506 on gastric mucosa in relation to the changes in NO synthase activity. METHODS: Gastric lesions were induced in rats by water immersion stress. Changes in NO synthase activity during water immersion stress treatment, and effects of FK506 on NO synthase activity were determined enzymatically. Gastric mucosal interleukin (IL)-1 beta and IL-2 were measured by immunoradiometric assay. Gastric mucosal blood flow was measured by hydrogen gas clearance technique. RESULTS: FK506 mitigated gastric lesions developed by water immersion stress. Stress-induced lesions were exacerbated by NG-monomethyl-L-arginine (L-NMMA), a specific inhibitor of NO synthase, while sodium nitroprusside, a NO donor, mitigated the lesions. Water immersion stress increased NO synthase activity in the early phase (0.5 h after stress treatment) and decreased it in the late phase (6 h after). Decrease in NO synthase activity in the late phase was significantly mitigated by FK506, though it did not affect changes in NO synthase activity in the early phase. Water immersion stress increased gastric mucosal IL-1 beta and IL-2 contents 6 h after stress treatment, and these increases were prevented by FK506. FK506 itself did not affect gastric mucosal blood flow. L-NMMA treatment significantly decreased gastric mucosal blood flow. In contrast, gastric mucosal blood flow was significantly increased by sodium nitroprusside. CONCLUSIONS: Increase in NO synthase activity might contribute to cytoprotection, and a decrease in activity might be a harmful factor for the gastric mucosa. Preservation of NO synthase activity by FK506 might be involved in FK506's protective effects on the gastric mucosa.

Induction of muscarinic cholinergic responsiveness in Xenopus oocytes by mRNA isolated from rat brain.

Microinjection of mRNA from adult rat brains into Xenopus oocytes induced an electrophysiological response to acetylcholine in the cells. The cells responded to acetylcholine through muscarinic receptors which appeared on the cell surface after microinjection of the mRNA and showed the depolarization, mainly due to an increase in membrane permeability to Cl ions. The acetylcholine reaction did not require extracellular Ca ions and was suppressed by pertussis toxin, suggesting the involvement of the inhibitory guanine nucleotide-binding regulatory protein.

Characterization of mRNA responsible for induction of functional sodium channels in Xenopus oocytes.

When Xenopus laevis oocytes were microinjected with poly(A)+ mRNA isolated from adult rat brains or electric organs of Electrophorus electricus, the oocytes developed functional sodium channels. Upon application of veratrine, the microinjected oocytes exhibited transient depolarization, resulting in spontaneous repetitive spikes in some occasions, and action potentials. These responses were mediated mainly by external Na ions, prolonged by scorpion toxin, completely blocked by tetrodotoxin, and suppressed by local anesthetics. Thus the mRNA-induced sodium channels exhibited essentially all the functional properties expected for native sodium channels in nerve and muscle membranes. Rat brain mRNA was fractionated into 4 fractions by sucrose gradient centrifugation. Each fraction and various combinations of them were examined for the efficiency in inducing functional sodium channels in Xenopus oocytes. A fraction corresponding to mRNA of approximately 30S to 46S was found to contain all mRNA necessary for the expression of the channels, indicating that mRNA of smaller sizes expected to code for smaller polypeptides may not be required.

[Clinical effectiveness of degradable starch microspheres (DSM) in patients with liver cancer].

Recently, degradable starch microspheres (DSM) have become available for use in patients with liver cancer in Japan. When DSM combined with a cytotoxic drug are infused through the hepatic artery, the steep drug concentration gradient to the tumor tissue results in a higher tissue drug concentration, which may elicit an increased antitumor response by blocking regional blood flow. Furthermore, the reduced systemic exposure of a coinjected drug can be translated into an increased regional extraction ratio due to blood flow reduction. DSM is infused via a catheter connecting to a subcutaneously implanted reservoir in outpatients. Pain is experienced by all patients. Other frequently observed adverse reactions are nausea and vomiting. However, these symptoms improve within a few hours. These observations indicate that intra-arterial chemotherapy combined with DSM may provide a more potent anticancer effect than a cytotoxic drug alone.

[Treatment of hemobilia after percutaneous transhepatic cholangiography and drainage (PTCD)].

PTCD was performed in 206 of our patients during the past 6 years and 7 months. Of the 206, hemobilia occurred in 14 patients (6.8%). The hemorrhage was completely stopped by irrigation of the bile duct in 3 patients, compression with a larger catheter in 7 patients, and transcatheter anterior embolization (TAE) in 4 patients. TAE was performed on the patients whose hemobilia could not controlled by the compression with a larger catheter. In TAE, either a steel coil or a sponge was used as an embolus. Rebleeding occurred in one patients for whom the right hepatic artery was chosen as a embolization site. Therefore, it was decided that the embolization was going to be done in all the hepatic arteries when the blood stream in the portal vein and preserved functions of the liver of the subjected patients including the one with rebleeding were fully normal. A complete control of the hemorrhage was obtained in all patients. The PTCD root caused hemobilia was removed after TAE in considering the possibility of rebleeding from the root, and a new PTCD root was made.

Endoscopic management and follow up of Dieulafoy lesion in the upper gastrointestinal tract.

BACKGROUND AND STUDY AIMS: Dieulafoy's lesion is an important cause of upper gastrointestinal bleeding, and the safety and efficacy of endoscopic treatment have been widely accepted. The aim of this study was to evaluate the effectiveness of endoscopic management, including hemoclipping and injection methods, for bleeding Dieulafoy lesions in the upper gastrointestinal tract. PATIENTS AND METHODS: Between 1995 and 2003, 61 patients with bleeding Dieulafoy lesions underwent endoscopic treatment. The available hemostatic methods were hemoclipping, hypertonic saline-epinephrine injection, and pure ethanol injection. Clinical data, endoscopic features, and treatment outcome were analyzed retrospectively. RESULTS: Comorbid conditions were present in 39 patients (64 %). Active bleeding was noted in 20 patients (33 %). Hemoclipping was a selected treatment in 48 patients (79 %). Initial hemostasis was achieved in 61 patients (100 %). One patient had rebleeding 6 days after the initial procedure but was successfully treated endoscopically. The 30-day mortality was 0 %. During follow-up, for a mean of 47 months, 15 patients (25 %) died of causes unrelated to the Dieulafoy lesion. Two patients had recurrent bleeding due to non-Dieulafoy gastric ulcer, and responded to endoscopic therapy. We encountered no patients who required surgery. CONCLUSIONS: Dieulafoy lesion can be successfully managed by endoscopic treatment. The long-term outcome is acceptable.

Acute haemorrhagic oedema of infancy associated with cytomegalovirus infection.

Acute haemorrhagic oedema (AHO) of infancy is a cutaneous leukocytoclastic vasculitis, clinically characterized by the acute development of peripheral oedema and targetoid purpuric lesions on the face and extremities. It usually affects children younger than 2 years of age. The disorder follows a benign course usually without recurrence or long-term complication. In most cases the origin is not clear, but underlying infections are assumed to play an aetiological role. We describe a 7-month-old boy whose clinical and histopathological features are typical of AHO. Serological tests clearly demonstrated a primary infection for cytomegalovirus (CMV). To our knowledge, this is the first reported case of AHO associated with CMV infection.

Etizolam-induced superficial erythema annulare centrifugum.

Erythema annulare centrifugum (EAC) is characterized by slowly enlarging annular erythematous lesions. Although the origin is not clear in most cases, EAC has been associated with infections, medications, and in rare cases, underlying malignancy. We describe a patient who developed annular erythematous lesions after etizolam administration. The eruptions were typical of the superficial form of EAC, both clinically and histopathologically. The lesions disappeared shortly after discontinuation of the medication. Patch testing with etizolam gave positive results. To our knowledge this is the first reported case of etizolam-induced superficial EAC.

Xanthoma disseminatum with large plaques confined to the back, pulmonary involvement and multiple intestinal xanthomas.

Xanthoma disseminatum (XD) is a rare benign mucocutaneous xanthomatosis that is classified as a benign non-Langerhans cell histiocytosis. We report a 68-year-old man who presented with peculiar, large plaques confined to the back 7 years after the onset of cranial diabetes insipidus. Histopathological features of the cutaneous lesions were typical of XD. The patient had lower respiratory tract involvement with histiocytic infiltrates, which was unresponsive to various treatments and resulted in a fatal outcome. Gastrointestinal endoscopies revealed multiple xanthomas in the sigmoid colon and the rectum. To our knowledge, this is the first reported case of intestinal xanthomas associated with XD.

Exacerbatory mechanism responsible for water immersion stress-induced gastric lesions in aged rats compared with young rats.

1. The present study was designed to investigate whether or not ageing affects the development of water immersion stress-induced gastric lesions in rats. Effects of cetraxate, an anti-ulcer drug, were also examined. 2. Gastric lesions were induced by 6 h water immersion stress in rats. Gastric mucosal blood flow was determined by the hydrogen gas clearance technique and nitric oxide synthase (NOS) activity was measured enzymatically. 3. Early development of gastric lesions was observed in aged rats and exacerbation of gastric lesions was also found. Lowering of gastric mucosal blood flow and reduced NOS activity were observed in aged rats. 4. Cetraxate mitigated the development of gastric lesions in young rats and also increased gastric mucosal blood flow and NOS activity. However, these favourable effects were diminished in aged rats. 5. Decreased NOS activity may be an important exacerbatory factor to the development of gastric lesions in aged rats. 6. Effects of cetraxate differed between young rats and aged rats. 7. These results may explain the refractoriness and drug resistance in gastric ulcers encountered by elderly individuals.