Periodontal tissue engineering by nano beta-tricalcium phosphate scaffold and fibroblast growth factor-2 in one-wall infrabony defects of dogs.

BACKGROUND AND OBJECTIVE: Nanoparticle bioceramics are being investigated for biomedical applications. We fabricated a regenerative scaffold comprising type I collagen and beta-tricalcium phosphate (ß-TCP) nanoparticles. Fibroblast growth factor-2 (FGF-2) is a bioeffective signaling molecule that stimulates cell proliferation and wound healing. This study examined the effects, on bioactivity, of a nano-ß-TCP/collagen scaffold loaded with FGF-2, particularly on periodontal tissue wound healing. MATERIAL AND METHODS: Beta-tricalcium phosphate was pulverized into nanosize particles (84 nm) and was then dispersed. A nano-ß-TCP scaffold was prepared by coating the surface of a collagen scaffold with a nanosize ß-TCP dispersion. Scaffolds were characterized using scanning electron microscopy, compressive testing, cell seeding and rat subcutaneous implant testing. Then, nano-ß-TCP scaffold, nano-ß-TCP scaffold loaded with FGF-2 and noncoated collagen scaffold were implanted into a dog one-wall infrabony defect model. Histological observations were made at 10 d and 4 wk postsurgery. RESULTS: Scanning electron microscopy images show that TCP nanoparticles were attached to collagen fibers. The nano-ß-TCP scaffold showed higher compressive strength and cytocompatibility compared with the noncoated collagen scaffold. Rat subcutaneous implant tests showed that the DNA contents of infiltrating cells in the nano-ß-TCP scaffold and the FGF-2-loaded scaffold were approximately 2.8-fold and 3.7-fold greater, respectively, than in the collagen scaffold. Histological samples from the periodontal defect model showed about five-fold greater periodontal tissue repair following implantation of the nano-ß-TCP scaffold loaded with FGF-2 compared with the collagen scaffold. CONCLUSION: The ß-TCP nanoparticle coating strongly improved the collagen scaffold bioactivity. Nano-ß-TCP scaffolds containing FGF-2 are anticipated for use in periodontal tissue engineering.

Bone augmentation using a highly porous PLGA/ß-TCP scaffold containing fibroblast growth factor-2.

BACKGROUND AND OBJECTIVE: Beta-tricalcium phosphate (ß-TCP), a bio-absorbable ceramic, facilitates bone conductivity. We constructed a highly porous three-dimensional scaffold, using ß-TCP, for bone tissue engineering and coated it with co-poly lactic acid/glycolic acid (PLGA) to improve the mechanical strength and biological performance. The aim of this study was to examine the effect of implantation of the PLGA/ß-TCP scaffold loaded with fibroblast growth factor-2 (FGF-2) on bone augmentation. MATERIAL AND METHODS: The ß-TCP scaffold was fabricated by the replica method using polyurethane foam, then coated with PLGA. The PLGA/ß-TCP scaffold was characterized by scanning electron miscroscopy (SEM), transmission electron microscopy (TEM), X-ray diffraction, compressive testing, cell culture and a subcutaneous implant test. Subsequently, a bone-forming test was performed using 52 rats. The ß-TCP scaffold, PLGA-coated scaffold, and ß-TCP and PLGA-coated scaffolds loaded with FGF-2, were implanted into rat cranial bone. Histological observations were made at 10 and 35 d postsurgery. RESULTS: SEM and TEM observations showed a thin PLGA layer on the ß-TCP particles after coating. High porosity (> 90%) of the scaffold was exhibited after PLGA coating, and the compressive strength of the PLGA/ß-TCP scaffold was six-fold greater than that of the noncoated scaffold. Good biocompatibility of the PLGA/ß-TCP scaffold was found in the culture and implant tests. Histological samples obtained following implantation of PLGA/ß-TCP scaffold loaded with FGF-2 showed significant bone augmentation. CONCLUSION: The PLGA coating improved the mechanical strength of ß-TCP scaffolds while maintaining high porosity and tissue compatibility. PLGA/ß-TCP scaffolds, in combination with FGF-2, are bioeffective for bone augmentation.

Application of collagen hydrogel/sponge scaffold facilitates periodontal wound healing in class II furcation defects in beagle dogs.

BACKGROUND AND OBJECTIVE: A three-dimensional scaffold may play an important role in periodontal tissue engineering. We prepared bio-safe collagen hydrogel, which exhibits properties similar to those of native extracellular matrix. The aim of this study was to examine the effect of implantation of collagen hydrogel/sponge scaffold on periodontal wound healing in class II furcation defects in dogs. MATERIAL AND METHODS: The collagen hydrogel/sponge scaffold was prepared by injecting collagen hydrogel, cross-linked to the ascorbate-copper ion system, into a collagen sponge. Class II furcation defects (of 5 mm depth and 3 mm width) were surgically created in beagle dogs. The exposed root surface was planed and demineralized with EDTA. In the experimental group, the defect was filled with collagen hydrogel/sponge scaffold. In the control group, no implantation was performed. Histometric parameters were evaluated 2 and 4 wk after surgery. RESULTS: At 2 wk, the collagen hydrogel/sponge scaffold displayed high biocompatibility and biodegradability with numerous cells infiltrating the scaffold. In the experimental group, reconstruction of alveolar bone and cementum was frequently observed 4 wk after surgery. Periodontal ligament tissue was also re-established between alveolar bone and cementum. Volumes of new bone, new cementum and new periodontal ligament were significantly greater in the experimental group than in the control group. In addition, epithelial down-growth was suppressed by application of collagen hydrogel. CONCLUSION: The collagen hydrogel/sponge scaffold possessed high tissue compatibility and degradability. Implantation of the scaffold facilitated periodontal wound healing in class II furcation defects in beagle dogs.

Root surface conditioning with bone morphogenetic protein-2 facilitates cementum-like tissue deposition in beagle dogs.

BACKGROUND AND OBJECTIVE: Modification of the root surface may play an important role in regenerating the periodontal attachment between the root and periodontal connective tissue. We speculated that bone morphogenetic protein (BMP) application to the root surface constructed a novel attachment by cementum-like hard tissue, although gingival connective tissue proliferated to the root surface. The aim of this study was to examine whether BMP-2 guided cementum-like tissue deposition on a BMP-conditioned root surface. MATERIAL AND METHODS: Root dentin on the buccal side of 24 teeth in four beagle dogs was surgically exposed. The denuded root dentin surfaces were demineralized with EDTA and washed with saline. Subsequently, 15 microL of BMP-2 solution (loading dose, 0.4 and 1.0 microg/microL) was applied to the root dentin surface. In the control roots, phosphate-buffered saline was applied to the root surface. Specimens were analyzed histologically 16 wk after surgery. RESULTS: Formation of cementum-like tissue was frequently observed on the BMP-2-conditioned root at the coronal portion. Cellular cementum-like tissue was separated from the original cementum and encapsulated with gingival connective tissue. Cementum-like tissue formation with BMP-2 at 1.0 microg/microL was significantly greater than that in the control roots and those with BMP-2 at 0.4 microg/microL. Downgrowth of the junctional epithelium in the 1.0 microg/microL BMP-2 group was significantly less than that in the control roots. CONCLUSION: Root dentin surface conditioning with BMP-2 stimulated cementum-like tissue formation and inhibited epithelial downgrowth.

Qualitative changes in the biologic characteristics of cultured fetal rat keratinizing epidermal cells during the process of malignant transformation after benzo[a]pyrene treatment.

In vitro malignant transformation of fetal rat keratinizing epidermal cells from inbred SD rats after benzo[a]pyrene (BP) treatment was analyzed from various biologic viewpoints. BP treatment directly and indirectly effected changes in cell growth characteristics, i.e., temperature dependence for growth, in vitro keratinization, chromosome structure, and the ability to form colonies on plastic substrate, on 0.57% agar medium layer, and in 0.33% soft agar medium. BP-treated cells at 30 degrees C remained in the premalignant stages and showed shifts in chromosome structure toward the hypodiploid range and parakeratotic changes in their keratinization process. However, the cells failed to form colonies even on a plastic substrate. BP-treated cell lines that adapted to temperatures of 35 and 37.5 degrees C remained in the premalignant stages; however, they acquired the ability to form colonies on plastic substrates during subcultivation. Malignantly transformed colonies appeared in these cell lines. In vitro keratinization processes were classified into nearly normal (diffuse lamellar, focal lamellar, and parakeratotic), intermediate, and atypical subtypes (columnar, spherical, and single-cell type). Cells of atypical keratinization subtypes and some of the intermediate subtypes formed squamous cell carcinomas in syngeneic hosts. Malignantly transformed cells showed shifts in chromosome structure toward the hypotetraploid range and colony formation on the 0.57% agar medium layer. However, they failed to form colonies in 0.33% soft agar medium. With the use of changes in biologic characteristics of the cells as indicators, fetal rat keratinizing epidermal cells in culture were classified into five stages. The appearance of stage III cells seemed to be the first key step in their malignant transformation.

Malignant transformation of a cloned, nontumorigenic mouse epidermal keratinocyte cell line, MSK-C3H-NU, by 7,12-dimethylbenz(a)anthracene.

MSK-C3H-NU, a cloned mouse epidermal keratinocyte cell line, was established from the epidermis of C3H/HeN mammary tumor virus-positive nude mice. Although it has lost its diploid chromosome number, the cell line is nontumorigenic, has been stable during serial subcultivations for over 2 years, and has retained some differentiated biological characteristics of normal keratinocytes. MSK-C3H-NU cells were cultured in growth medium containing 7,12-dimethylbenz(a)anthracene. After 2 months, colonies exhibited marked changes in cell morphology, cell arrangement, and keratinization pattern that appeared. The transformation frequency per 10(5) survivors in 7,12-dimethylbenz(a)anthracene-treated (10, 100, and 500 ng/ml) subgroups was 0, 119, and 1370 for Experiment I and 3.9, 238, and 2500 for Experiment II, respectively. Most of these transformed cells became malignant and formed tumors in nude mice. Histologically, the tumors were well-differentiated, keratinizing, squamous cell carcinomas showing papillary growths. In 7,12-dimethylbenz(a)anthracene-treated subgroups, cells from colonies that retained the original morphological characteristics did not form tumors in animals, and in control groups, no cell population showed tumorigenicity. In the MSK-C3H-NU cell system, the morphological alterations seem to be strongly associated with malignant conversion.

Reversible induction of anchorage independent growth from normal mouse epidermal keratinocytes, MSK-C3H-NU, in soft agar medium by 12-O-tetradecanoylphorbol-13-acetate and epidermal growth factor.

The induction of anchorage independent growth occurred in nontumorigenic, mouse epidermal keratinocyte, MSK-C3H-NU cells when inoculated in a 0.3% soft agar medium with 12-O-tetradecanoylphorbol-13-acetate and/or epidermal growth factor. Colonies appeared about 3 weeks after incubation and keratinization of various forms occurred in them. 10-43 cells, a subline of higher subcultivation level, showed a rather sensitive response to these chemicals. 12-O-tetradecanoylphorbol-13-acetate and epidermal growth factor each induced anchorage independent growth but additive effects were also observed. Cell lines were established from clonal growths recovered from these induced colonies in soft agar. Morphologically, they retained their original normal clonal growth and keratinization patterns. Anchorage independent growth was not retained. Response to 12-O-tetradecanoylphorbol-13-acetate and epidermal growth factor, however, became more sensitive in some of the recovered cell lines when compared to their original cells. These data show that the induction of anchorage independent growth in soft agar is reversible in the MSK-C3H-NU cell system.

Changes in expressions of proteasome and ubiquitin genes in human renal cancer cells.

Proteasomes and ubiquitin (Ub) are essential components of the energy-dependent, nonlysosomal proteolytic pathway. To clarify the physiological role of this proteasome/Ub-dependent pathway, we meaured the levels of expressions of proteasomes and Ub in human renal cancers by Northern blot and immunochemical analyses. The mRNAs for two of the multiple subunits of proteasomes, C2 and C9, were expressed at abnormally high levels in most neoplastic lesions of patients with various primary renal cell carcinomas and in all renal cancer cell lines examined. However, no significant difference was found by enzyme immunoassay in the proteasomal contents of cancerous and normal parts of the kidney. The levels of mRNAs for the subunits of proteasomes were high in rapidly proliferating renal cells and appeared to be correlated with the activities of these cells for proteasome synthesis, but the cellular contents of proteasomes in these cells were normal, suggesting rapid turnover of proteasomes in rapidly proliferating cancer cells. Consistent with the increased expressions of proteasomal mRNAs, the expressions of three Ub genes, mono-UbA80, mono-UbA52, and poly-UbC, were found to be greatly increased in these renal cancer cells. Immunohistochemical staining of normal kidney showed that the levels of both proteasomes and Ub were high in cells of renal tubules and collecting ducts, but low in the glomerulus. The levels of both proteins appeared to be considerably increased in the nuclei of granular and clear carcinoma cells of the kidney. Moreover, the profiles of cellular proteins conjugated with Ub in normal kidney tissues were different from those in cancerous parts of the kidney and in established renal cancer cells. These results suggest that the proteasome- and ubiquitin-mediated system is functionally involved in the cancerous state in human kidney.

Semaphorin 3A Induces Odontoblastic Phenotype in Dental Pulp Stem Cells.

In cases of pulp exposure due to deep dental caries or severe traumatic injuries, existing pulp-capping materials have a limited ability to reconstruct dentin-pulp complexes and can result in pulpectomy because of their low potentials to accelerate dental pulp cell activities, such as migration, proliferation, and differentiation. Therefore, the development of more effective therapeutic agents has been anticipated for direct pulp capping. Dental pulp tissues are enriched with dental pulp stem cells (DPSCs). Here, the authors investigated the effects of semaphorin 3A (Sema3A) on various functions of human DPSCs in vitro and reparative dentin formation in vivo in a rat dental pulp exposure model. Immunofluorescence staining revealed expression of Sema3A and its receptor Nrp1 (neuropilin 1) in rat dental pulp tissue and human DPSC clones. Sema3A induced cell migration, chemotaxis, proliferation, and odontoblastic differentiation of DPSC clones. In addition, Sema3A treatment of DPSC clones increased ß-catenin nuclear accumulation, upregulated expression of the FARP2 gene (FERM, RhoGEF, and pleckstrin domain protein 2), and activated Rac1 in DPSC clones. Furthermore, in the rat dental pulp exposure model, Sema3A promoted reparative dentin formation with dentin tubules and a well-aligned odontoblast-like cell layer at the dental pulp exposure site and with novel reparative dentin almost completely covering pulp tissue at 4 wk after direct pulp capping. These findings suggest that Sema3A could play an important role in dentin regeneration via canonical Wnt/ß-catenin signaling. Sema3A might be an alternative agent for direct pulp capping, which requires further study.

Formation of 5alpha-reduced C19 steroids from progesterone in vivo by 5alpha-reduced pathway in older prepubertal rat testis.

Either [3H] progesterone (0.5 or 5 nmol/5 muCi), 5alpha-[3H] pregnane-3,20-dione (5 nmol/5 muCi) or [14C] progesterone (6.6 nmol/0.2 muCi) plus 5alpha-[3H]-pregnane-3,20-dione (1 or 6.6 nmol/0.6 muCi), suspended in 0.05 ml of physiological saline solution, was injected into each testis of 32- and 90-day-old rats. Following injection, radioactive metabolites in testis and spermatic vein blood were extracted, isolated, measured and identified by column and paper chromatographies, with derivative formation and recrystallization to constant specific activity. In the blood and testis of older prepubertal rats, major 17-OH-C21 and C19 metabolites of progesterone were 5alpha-reduced steroids such as 3alpha, 17alpha-dihydroxy-5alpha-pregnan-20-one, 5alpha-androstane-3alpha,17beta-diol and androsterone. Following injection of [14C] progesterone plus 5alpha-[3H] pregnane-3,20-dione into 32-day-old rat testis, no significant augmentation of the isotope from progesterone was observed in 5alpha-reduced C19 steroids as compared with 5alpha-reduced 17-OH-C21 steroids, indicating that 5alpha-reduced C19 steroids were mainly formed from 5alpha-reduced 17-OH-C21 steroids in older prepubertal testis. In the blood and testis of adult rats, small amounts of 5alpha-reduced metabolites were shown to be produced from progesterone, while active 17alpha-hydroxylation of 5alpha-pregnane-3,20-dione followed by C17-C20-lyase reaction was demonstrated. These findings seem to indicate that formation of 5alpha-reduced C19 steroids from progesterone by the 5alpha-reduced pathway is a major pathway of androgen biosynthesis in older prepubertal rat testis in vivo.

Clinical outcomes of tracheoesophageal diversion for intractable aspiration.

OBJECTIVES: The purpose of the present study was to examine the clinical outcomes of using tracheoesophageal diversion for preventing intractable aspiration. METHOD: We retrospectively reviewed 25 patients who underwent tracheoesophageal diversion from 2003 to 2009 at our hospital (median age, 25 years; range, 0-78 years). End-to-side anastomosis was used in 16 cases and side-to-side anastomosis was used in 9. RESULTS: The average operative time was 141 minutes for end-to-side anastomosis and 191 minutes for side-to-side anastomosis. Peri-operative complications were observed in only two (8 per cent) cases: one with infection and one with haematoma. No fistulas were observed. Aspiration was prevented in all cases, but the nutritional route depended on the swallowing function of the patient. Oral feeding was the main nutritional route after surgery in only four patients (16 per cent). CONCLUSION: This procedure is well suited to patients who lack speech communication and are at high risk of aspiration.

Pharmacokinetics of clarithromycin in Helicobacter pylori eradication therapy in patients with liver cirrhosis.

BACKGROUND: Proton pump inhibitor triple therapy with clarithromycin and metronidazole has been widely used for Helicobacter pylori eradication. However, the efficacy and the safety of this therapy in patients with liver cirrhosis have not been established. AIM: To evaluate the effect of hepatic dysfunction on metabolism of clarithromycin as it is used for H. pylori eradication therapy in patients with liver cirrhosis, and the efficacy of eradication therapy in those patients. METHODS: Serum levels of clarithromycin and its metabolite. 14-(R)-hydroxyclarithromycin, were examined in 18 subjects (five normal controls and 13 hospitalized patients with liver cirrhosis) on a selected day between days 7 and 10 of a 2-week course of eradication therapy. This therapy consisted of lansoprazole (30 mg, once a day) together with clarithromycin (200 mg, twice a day) and metronidazole (250 mg, twice a day). In addition, 118 H. pylori-positive out-patients, 88 with peptic ulcer and 30 with liver cirrhosis, underwent the same eradication therapy. RESULTS: Values for the area under the 0-6 h concentration-time curve (AUC) for clarithromycin were not significantly different among the groups. However, the AUC (0-6 h) values of 14-(R)-hydroxyclarithromycin were significantly lower in the Child-Pugh C group than in either the normal controls or the Child-Pugh A/B group. The cure rate for the peptic ulcer patients was 84% on a per protocol analysis (95% CI: 80%-88%) and 81% on an intention-to-treat analysis (95% CI: 77%-85%), while in the liver cirrhosis patients it was 89% in a per protocol analysis (95% CI: 78%-99%) and 83% in an intention-to-treat analysis (95% CI: 70%-97%). Mild adverse effects were observed in 10% of the peptic ulcer patients and 13% of the liver cirrhosis patients, with none leading to premature withdrawal from the study. CONCLUSION: The 2-week low-dose lansoprazole-based triple therapy tested is a simple, effective and well-tolerated regimen for H. pylori eradication in patients with liver cirrhosis.

Susceptibility of Helicobacter pylori isolates to metronidazole, clarithromycin and amoxycillin in vitro and in clinical treatment in Japan.

BACKGROUND: Primary and acquired resistance to antibiotics is an important factor in determining the reason for treatment failure in Helicobacter pylori infection. We examined the relationship between the susceptibility of H. pylori isolates and the efficacy of chemotherapy. METHODS: The minimal inhibitory concentrations (MICs) of metronidazole (MNZ), clarithromycin (CLAR) and amoxycillin (AMOX) of 320 H. pylori pre-treatment isolates were determined by the agar dilution method. In 290 patients with peptic ulcers. H. pylori infection was treated by dual or triple combination therapies for 2 weeks: one proton pump inhibitor (30 mg/day lansoprazole or 20 mg/day omeprazole) and one or two antibiotics (500 mg AMOX, 200 mg CLAR or 250 mg MNZ twice a day). MICs were also determined after the treatment failure. RESULTS: Among the drugs tested, for MNZ and CLAR, 8.1% and 9.1% of the isolates, respectively, were resistant, while no isolate was resistant to AMOX. After unsuccessful treatment using MNZ and CLAR, 66.7% and 70.61% of the isolates changed from sensitive to resistant, respectively. All isolates were sensitive to AMOX after treatment failure. CONCLUSIONS: The failure of the H. pylori treatment results in the induction of resistance to CLAR and/or MNZ. Regimens with a high cure rate should be used in order to prevent the generation of acquired resistance to antibiotics.

The effect of helicobacter pylori eradication therapy on gastric antral myoelectrical activity and gastric emptying in patients with non-ulcer dyspepsia.

BACKGROUND: Dysmotility of the gastroduodenal region and delayed gastric emptying have been considered to play roles in non-ulcer dyspepsia. In addition, it has been reported that Helicobacter pylori induced inflammation of the gastric mucosa may affect gastric motility. AIM: To evaluate the effects of H. pylori eradication therapy on gastrointestinal motility and symptoms in non-ulcer dyspepsia patients. METHODS: A total of 46 non-ulcer dyspepsia patients were examined for gastric emptying, antral myoelectrical activity, H. pylori infection, and symptom scores. In H. pylori-positive non-ulcer dyspepsia patients, gastric emptying, antral myoelectrical activity, and symptom scores were also analysed 2 months after being cured of H. pylori infection. RESULTS: A total of 67.4% of the non-ulcer dyspepsia patients were H. pylori-positive. Both abnormal gastric emptying and antral myoelectrical activity were observed in non-ulcer dyspepsia patients. H. pylori-positive non-ulcer dyspepsia patients were divided into three groups according to their gastric emptying: the delayed gastric emptying group, the normal gastric emptying group, and the rapid gastric emptying group. In the delayed and rapid gastric emptying groups, the gastric emptying and symptom scores were improved significantly by the eradication therapy. However, there was no improvement in symptom scores in the normal gastric emptying non-ulcer dyspepsia group by the eradication therapy. CONCLUSIONS: Disturbed gastric emptying and antral myoelectrical activity play roles in non-ulcer dyspepsia. Helicobacter pylori infection, inducing disturbed gastric emptying, may cause some non-ulcer dyspepsia symptoms. Gastric emptying and symptom scores are improved by H. pylori eradication therapy in non-ulcer dyspepsia patients with disturbed gastric emptying. H. pylori eradication therapy is effective in H. pylori-positive non-ulcer dyspepsia patients with disturbed gastric emptying.

The effect of Helicobacter pylori eradication therapy on gastric antral myoelectrical activity and gastric emptying in patients with non-ulcer dyspepsia.

BACKGROUND: Dysmotility of the gastroduodenal region and delayed gastric emptying have been considered to play roles in non-ulcer dyspepsia (NUD). Helicobacter pylori-induced inflammation of the gastric mucosa may affect gastric motility. AIM: To evaluate the effects of H. pylori eradication therapy on gastrointestinal motility and symptoms in NUD patients. METHODS: : Forty-six NUD patients were examined for gastric emptying, antral myoelectrical activity, H. pylori infection, and symptom scores. In H. pylori-positive NUD patients, gastric emptying, antral myoelectrical activity, and symptom scores were also analysed 2 months after cure of H. pylori infection. RESULTS: Sixty-seven per cent of NUD patients were H. pylori-positive. Both abnormal gastric emptying and antral myoelectrical activity were observed in NUD patients. H. pylori-positive NUD patients were divided into three groups according to their gastric emptying: the delayed group, the normal group, and the rapid group. In the delayed and rapid gastric emptying groups, the emptying and symptom scores were improved significantly by eradication. There was no improvement in symptom scores in the normal gastric emptying NUD group by the eradication therapy. CONCLUSIONS: Disturbed gastric emptying and antral myoelectrical activity play roles in NUD. H. pylori-induced disturbed gastric emptying may cause some NUD symptoms. Gastric emptying and symptom scores are improved by H. pylori eradication therapy in NUD patients with disturbed gastric emptying; H. pylori eradication therapy is effective in H. pylori-positive NUD patients with disturbed gastric emptying.

Evaluation of endoscopic 13C-urea breath test for assessment of Helicobacter pylori eradication.

To minimize the inaccuracies of the diagnostic methods for Helicobacter pylori infection, we developed a new diagnostic method and called it the endoscopic 13C-urea breath test (EUBT). We evaluated the accuracy of EUBT for detecting this infection and assessing its eradication. EUBT was conducted on 267 patients with gastroduodenal disease. After the collection of a baseline breath sample, gastroduodenal endoscopy was performed. A 20-ml aliquot of a solution containing 100 mg of 13C-urea was sprayed over the entire gastric mucosa via the endoscope. A breath sample was then collected 15 min after spraying. The content of 13CO2 was measured in the breath samples by ratio mass spectrometry. Two biopsy specimens each from the antrum and the middle corpus were obtained for culture and histology. The EUBT cutoff level was 1.4%. The sensitivity and specificity of EUBT for the diagnosis of H. pylori infection were both 98.2%, and for assessment of the eradication they were 100% and 98.9%, respectively. EUBT is an accurate method not only for the diagnosis of H. pylori infection but also for assessment of its eradication.

Immunogenetic analysis of the human leukocyte antigen DQA1 locus in patients with duodenal ulcer or chronic atrophic gastritis harbouring Helicobacter pylori.

AIM: To examine the human leukocyte antigen (HLA)-DQA1 locus in patients harbouring Helicobacter pylori with chronic atrophic gastritis or duodenal ulcer as part of an investigation into immunogenetic differences in the host. SUBJECTS AND METHODS: We examined 116 patients harbouring H. pylori (55 patients with chronic atrophic gastritis, 61 with duodenal ulcers) and 28 H. pylori-negative healthy controls for HLA-DQA1 genotypes. H. pylori infection was determined by culturing biopsy samples from the gastric body and antrum and by enzyme-linked immunosorbent assay. HLA-DQA1 typing was carried out by the polymerase chain reaction-restriction fragment length polymorphism method. RESULTS: The allele frequency of DQA1*0102 was significantly higher in H. pylori-negative controls (0.250) than in H. pylori-positive duodenal ulcer patients (0.090). In contrast, the allele frequency of DQA1*0301 was significantly lower in H. pylori-negative controls (0.214) than in H. pylori-positive duodenal ulcer patients (0.418). CONCLUSION: These results suggest that there are genetic differences in the HLA-DQA1 locus between H. pylori-positive duodenal ulcer patients and H. pylori-negative healthy controls.

Studies on interrelation of structure and antitumor effects of polysaccharides: antitumor action of periodate-modified, branched (1 goes to 3)-beta-D-glucan of Auricularia auricula-judae, and other polysaccharides containing (1 goes to 3)-glycosidic linkages.

Antitumor activities of two (1 goes to 3)-beta-D-glucans, isolated from the fruiting body of Auricularia auricula-judae ("kikurage", an edible mushroom), and other branched polysaccharides containing a backbone chain of (1 goes to 2)-alpha-D-glucosidic or (1 goes to 3)-alpha-D-mannosidic linkage [and their corresponding (1 goes to 3)-D-glycans, derived by mild, Smith degradation] were compared. Among these polysaccharides, a water-soluble, branched (1 goes to 3)-beta-D-glucan (glucan I) of A. auricula-judae exhibited potent, inhibitory activity against implanted Sarcoma 180 solid tumor in mice. The alkali-insoluble, branched (1 goes to 3)-beta-D-glucan (glucan II), a major constituent of the fruiting body, showed essentially no inhibitory activity. When the latter glucan, having numerous branches attached, was modified by controlled, periodate oxidation, borohydride reduction, and mild, acid hydrolysis, the resulting, water-soluble, regraded glucan, having covalently linked polyhydroxy groups attached at O-6 of the (1 goes to 3)-linked D-glucosyl residues, exhibited potent antitumor activity. Further investigations using the glucan-polyalcohol indicated that the attachment of the polyhydroxy groups to the (1 goes to 3)-beta-D-glucan backbone may enhance the antitumor potency of the glucan. On the other hand, partial introduction of carboxymethyl groups into glucan II (d.s., 0.47--0.86), which altered the insolubility property, failed to enhance the antitumor activity. The interrelation between the antitumor activity and the structure of the branched (1 goes to 3)-beta-D-glucan is discussed, on the basis of methylation and 13C-n.m.r. studies of the periodate-modified glucans.

Structure of pestalotan, a highly branched (1----3)-beta-D-glucan elaborated by Pestalotia sp. 815, and the enhancement of its antitumor activity by polyol modification of the side chains.

Pestalotia sp. 815, a newly isolated fungus, produces extracellularly a highly (1----6)-branched (1----3)-beta-D-glucan in high yield when grown in a D-glucose-containing medium. This extracellular glucan, designated "Pestalotan", has [alpha]25D-0.1 degree (c 0.5, M NaOH) and a molecular weight greater than 2 X 10(6). Chemical and enzymic studies indicated that pestalotan has a very highly branched structure containing a back-bone chain of beta-D-(1----3)-linked D-glucosyl residues, and three out of five D-glucosyl residues are substituted at O-6, mostly with single D-glucosyl groups, and a very few with short beta-(1----6)-linked oligosaccharide units. This D-glucan becomes water-insoluble after isolation from the culture filtrate followed by dehydration, and shows moderate growth-inhibitory activities against mouse-implanted tumors. However, when the D-glucosyl groups of the side chains were modified by periodate oxidation and borohydride reduction, the resulting, still water-insoluble, D-glucan polyol exhibited potent antitumor activities, confirming that the attachment of many polyhydroxy groups to the beta-D-(1----3)-linked D-glucan back-bone gives a remarkable enhancement effect on the antitumor activity of the branched D-glucan. Prolonged treatment of the D-glucan polyol by ultrasonic irradiation afforded a low-molecular-weight D-glucan polyol (SD-pestalotan polyol), without alteration of its chemical structure. The water-soluble, SD-pestalotan polyol, having a molecular weight of 4.7 X 10(5), exhibited remarkable antitumor activities against both allogeneic and syngeneic, mouse-implanted tumors, at small dosages (1-5 mg/kg for 10 days) by intraperitoneal administration. A comparison of values of the molecular weight of SD-pestalotan polyol, estimated by 3-MPa l.c. for the aqueous solution and the dimethyl sulfoxide solution, strongly suggested that the D-glucan polyol must form a triple-chain conformation in aqueous solution.

Selective inhibition of IL-2 gene expression by trichostatin A, a potent inhibitor of mammalian histone deacetylase.

During screening for inhibitors of T cell activation, we have found that trichostatin A (TSA), known as a potent inhibitor of histone deacetylase, showed selective inhibitory activity against IL-2 gene expression. From luciferase reporter experiments on human leukemic Jurkat T cells, TSA was found to inhibit the expression of the luciferase reporter gene directed by the IL-2 enhancer and promoter with a 50% inhibitory concentration value of 0.073 microM. On the other hand, TSA, at the same concentration, enhanced the expression of the luciferase reporter gene directed by the c-fos enhancer and promoter. The result of RT-PCR experiments also indicates that TSA has selective inhibitory activity against IL-2 gene expression in Jurkat cells. These results suggest that the change in chromatin structure caused by the hyperacetylation of histone might affect the regulation of IL-2 and c-fos gene expression.