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Biomedical subjects

Toshikazu Yamaguchi

Publications and source records attributed to Toshikazu Yamaguchi.

17 recordsLinked to original sources

Classification of hepatitis B virus genotypes by the PCR-Invader method with genotype-specific probes.

Hepatitis B virus is a worldwide public health problem. A simple and effective test to identify viral genotypes would greatly aid efforts to understand and control the spread of this disease. A serial invasive signal amplification reaction assay (PCR-Invader assay) was developed for distinguishing the known eight genotypes (A-H) and four subgenotypes (Aa, Ae, Ba, Bj) of hepatitis B virus (HBV). The preS/S and core regions were amplified by multiplex PCR and delivered to 12 wells containing genotype-specific Invader probes. By observing the fluorescence patterns in the wells, HBV sub/genotypes can be assigned. A total of 505 serum samples containing HBV/HBsAg in Japan was examined by PCR-Invader and compared the results with those from ELISA assays with monoclonal antibodies against epitopes on gene products of the preS2 region and with a genotype-specific probe assay (GSPA) based on the preS1 region. Genotypes determined by the PCR-Invader agreed with those of the ELISA method in 98.2% of cases and with the GSPA method in 97.1% of cases. Co-infection with two distinct genotypes was correctly identified by the PCR-Invader in four serum samples, as determined by GSPA. Thus, the PCR-Invader assay is a useful tool for detecting the 10 known HBV sub/genotypes.

Base Sequence↗

Evaluation of the state of active ingredients in pharmaceutical preparations using fourier transform-Raman difference spectra.

To examine the pharmaceutical application of Fourier transform (FT)-Raman spectroscopy, the state of active pharmaceutical ingredients (APIs) in a preparation of several forms was evaluated by investigating the Raman difference spectra between the preparation and excipient. The difference spectra indicated that APIs in alacepril tablets, caffeine sustained-release granules, and quinidine sulfate granules remained unchanged after the manufacturing process. However, the state of sparfloxacin in nanoparticles changed, although it remained unchanged in tablets or powders. These results show that the FT-Raman difference spectrum is expected to be utilized in the field of quality control of crystalline pharmaceutical preparations.

Angiotensin-Converting Enzyme Inhibitors↗

Significance of liver negative-strand HCV RNA quantitation in chronic hepatitis C.

BACKGROUND/AIMS: Liver negative-strand hepatitis C virus (HCV) RNA is the most direct indicator of active viral replication but has only been examined in a few semiquantitative studies. METHODS: Positive- and negative-strand HCV RNA in the right (R) and left (L) liver lobes was quantified by rTth-based strand-specific real-time polymerase chain reaction for 48 chronic hepatitis C patients. RESULTS: Close correlations between lobes were seen for positive- and negative-strand amounts (r = 0.950; P < 0.001 and r = 0.920; P < 0.001, respectively). The ratio of negative to positive strands (median, 0.14 for R and 0.13 for L) varied by 2 log directly in relation to HCV replication assessed by liver negative strands but had no relation to liver positive strands and circulating HCV. Only negative-strand quantitation was inversely correlated with age (r = -0.322; P = 0.026 for R and r = -0.340; P = 0.018 for L), while liver tissues with hepatitis B virus DNA contained larger amounts of each strand. In 27 patients treated with enhanced interferon monotherapy, the amounts of liver negative strands (<4 log copies/100 ng RNA) were the only independent predictor of a sustained virologic response. CONCLUSIONS: Negative-strand quantitation is uniform in the liver and bears distinct relevance to the disease.

Adult↗

Mucin phenotypic expression and p53 gene abnormality of gastric super-minute well-differentiated adenocarcinoma: re-evaluation with relationship between histogenesis of well-differentiated adenocarcinoma and intestinal metaplasia in distal stomach.

BACKGROUND: Although the gastric well-differentiated adenocarcinoma in the distal stomach has been thought to develop via a intestinal metaplasia-carcinoma sequence, there are some disproofs from new mucin examinations for minute-size lesions in same type carcinoma. The current study was performed and pointed out the new findings for the solution to the problem according to the point described above. METHODS: 12 super-minute lesions (less than 1 mm in maximum diameter) of well-differentiated adenocarcinoma in distal stomach (SMCa), which were detected from the pathological examinations of 210 surgically resected stomach specimens, and the mucosa adjacent to these carcinoma lesions, were examined by immunohistochemical mucin stainings (MUC2 and CD-10: intestinal phenotype, 45M1 and MUC6: gastric phenotype) and p53-overexpression. And the analyses of the replication error of the microsatellites in chromosome 17 related p53 gene (TP53 and D17S786) (RER-p53MS) were performed in SMCa lesions, adjacent mucosa to each lesion and other gastric mucosa with intestinal metaplasia, because all SMCa lesions showed p53-overexpression immunohistochemically, described below. RESULTS: 1. The carcinoma cells in all SMCa lesions were positive for 45M1 and p53. On the other hand, no positive carcinoma cells for MUC6 were seen although the pyloric glands and the remnant pyloric gland in the SMCa lesions in the same slides were positive for MUC6. Ten lesions (83%) had intestinal phenotypic mucin (10 lesions: MUC2 (+), 4 lesions: CD10 (+)). Two lesions (17%) were positive for only 45M1 (gastric phenotypic mucin). 2. All of the mucosa adjacent to SMCa showed intestinal metaplasia (complete type: 7 regions, incomplete type: 5 regions). 3. RER-p53MS was confirmed in 42% (5/12 regions) of SMCa, in 42% (5/12 regions) of the mucosa adjacent to SMCa and 14% (6/42 regions) of the other intestinal metaplasia mucosa. CONCLUSION: Most of the super-minute well-differentiated adenocarcinoma lesions in the distal stomach, which had both gastric and intestinal phenotypic mucin, are considered to develop from the tubular proliferative zone with the incomplete type of the intestinal metaplasia and p53 gene abnormality, while a part of them, which had only gastric phenotypic mucin, may derive from the gastric native tubules (non-metaplastic epithelium) with p53 gene abnormality.

Journal Article↗

Intercalated duct cell is starting point in development of pancreatic ductal carcinoma?

BACKGROUND: Although it is well known that the pancreatic ductal carcinoma may develop having a relationship to the mucous gland hyperplasia (MGH) with atypia (PanIN-1B by PanIN system), the starting point of this atypical MGH is unclear. To know it, we examined the pancreas tissue using many methods described below. METHODS: 1. Twenty-seven surgically resected pancreas tissue specimens, including pancreatic ductal carcinomas (PDC), chronic pancreatitis and normal pancreas, were investigated using immunohistochemical stainings for MUC1, MUC6, 45M1, Ki67 and p53. 2. DNA extraction and analysis of K-ras mutation at codon 12 using microdissection method: The paraffin blocks with 16 regions including the intercalated duct cell (IC) adjacent to the atypical MGH were prepared for DNA extraction. Mutation of K-ras codon 12 was analyzed and compared in enriched polymerase chain reaction-enzyme-linked mini sequence assay (PCR-ELMA). RESULTS: 1. In the normal pancreas, although no positive cell was seen in 45M1, p53, Ki67, the cytoplasm of IC were always positive for MUC1 and sometimes positive for MUC6. In the pancreas with fibrosis or inflammation, MGH was positive for MUC6 and 45M1. And atypical MGH was positive for MUC1, MUC6 and 45M1. Some IC adjacent to the atypical MGH was positive for Ki67 as well as atypical MGH. The carcinoma cells in all cases of PDC were diffusely positive for MUC1, 45M1, p53 and Ki67, and focally positive for MUC6. 2. In K-ras mutation, we examined the regions including IC adjacent to the atypical MGH, because the immunohistochemical apomucin stainings of these regions resembled those of PDC as described above. And K-ras mutation was confirmed in 12 of 16 regions (75%). All mutations were a single mutation, in 6 regions GTT was detected, in 4 regions GAT was detected and in 2 region AGT was detected. CONCLUSION: Some intercalated duct cell may be the starting point of the pancreatic ductal carcinoma, because the exhibitions of mucin expressions, Ki67, p53 and K-ras mutation in some intercalated duct cell resembled those of mucous gland hyperplasia or pancreatic ductal carcinoma.

Journal Article↗

Colonic Paneth cell metaplasia is pre-neoplastic condition of colonic cancer or not?

ABSTRACTS: BACKGROUND: The carcinogenesis of colorectal cancer has been accepted by a model for a cascade of genetic alterations, named the adenoma-carcinoma sequence. In order to elucidate the carcinogenesis of the colorectal cancer more clearly, the genetic abnormalies of the non-neoplastic mucosal epithelium of the colon and rectum should be investigated. It has been speculated that colonic Paneth cell metaplasia (PaM) is one of the pre-neoplastic mucosa of colonic cancer. Therefore, we studied the propria mucosa of the right colon with PaM from the standpoints of the frequency of the K-ras codon 12 mutations (K-ras), which is initial genetic abnormality in colorectal cancer, and the loss of heterozygosity of microsatellite markers (LOH-MS), which has a relationship to development of colorectal cancer. METHODS: Fifty-two regions with PaM histopathologically from 12 surgically resected right colon specimens were studied. DNA extraction of the colonic mucosa with PaM was obtained using a microdissection method, and the frequency of the K-ras of PaM was investigated by enriched polymerase chain reaction-enzyme linked mini-sequence assay, and the frequency of the LOH-MS (D2S123, D17S250 and D5S346) of PaM was examined by high resolution fluorescenced labeled PCR primers. RESULTS: K-ras mutation was detected in fifteen regions among 52 PaM (28.9%). All mutations were a single mutation and GGT changed to AGT in eleven and GAT in four. LOH-MS were detected in twenty-one regions among 52 PaM (40.4%) (D2S123: 35.4%, 17/48 regions, D17S250: 13.7%, 7/51 regions, and D5S346: 0%, 0/52 regions). No K-ras mutations and LOH-MS were detected in the controls (Colorectal mucosa with no PaM). CONCLUSIONS: Colonic mucosa with Paneth cell metaplasia may be one of the pre-neoplastic mucosa in the development of the colonic epithelial neoplasia.

Journal Article↗

Design of controlled-release morphine suppositories containing polyglycerol ester of fatty acid.

Controlled-release morphine suppositories were prepared by utilizing polyglycerol ester of fatty acid (PGEF). The addition of PGEF to fatty suppository base Witepsol H15 resulted in a decrease of morphine release rate from suppositories. Among PGEFs examined, decaglycerol heptabehenate (HB750) was the most effective additive for the controlled-release of morphine from fatty suppositories. The apparent viscosity of suppository bases increased with the increase in HB750 content, and good correlation was observed between the apparent viscosity of suppository bases at 37 degrees C and the amount of HB750 added in the mixed base. The in vitro release rate of morphine was decreased by the addition of HB750 and the release rate constant (Higuchi's rate constant) for morphine release was significantly correlated with the HB750 content in the mixed bases as well as the apparent viscosity of mixed bases, indicating that the release of morphine from the mixed bases could be regulated by the HB750 content in the mixed bases. After rectal administration of Witepsol H-15-HB750 mixed suppositories to dogs, plasma concentrations of morphine did not increase rapidly at early time periods, but relatively high levels of morphine in plasma were sustained for longer time periods. Mean residence time of morphine was considerably prolonged without changing relative bioavailability in the case of the mixed base suppositories containing 15-17% HB750, compared with the Witepsol H15 suppository, clearly indicating that the mixed bases containing HB750 are expected to be useful for the design of controlled-release morphine suppositories.

Analgesics, Opioid↗

Pathologic changes in the non-carcinomatous epithelium of the gallbladder in patients with a relatively long common channel.

BACKGROUND: Except for pancreaticobiliary maljunction, the relationship between a relatively long common channel and gallbladder carcinoma is unknown. METHODS: For purposes of this study, a high confluence of pancreaticobiliary ducts was defined as a common channel that is 6 mm or greater in length, together with occlusion of the communication between the pancreatic and bile ducts during sphincter contraction. Pancreaticobiliary maljunction and high confluence of the pancreaticobiliary ducts were detected in 69 (2.1%) and 54 (1.6%), respectively, of 3300 consecutive patients who underwent ERCP. Proliferation activity and genetic alteration were examined in the non-carcinomatous epithelium of the gallbladder in patients with these two radiographic findings at ERCP. RESULTS: The Ki-67 labeling index in the epithelium in cases of a high confluence of the pancreaticobiliary ducts and pancreaticobiliary maljunction without biliary dilatation was significantly greater than that in cases of gallbladder carcinoma without these anomalies (p < 0.01). Overexpression of p53 and K-ras mutations were detected in, respectively, 22.2% and 27.8% of cases of a high confluence of pancreaticobiliary ducts. CONCLUSIONS: A relatively long common channel may be an important risk factor for the development of gallbladder carcinoma. Vigilance for the development of gallbladder carcinoma is indicated in patients with a relatively long common channel, in addition to those with pancreaticobiliary maljunction.

Bile Ducts↗

Occult lymphoma cells prevalent in autologous marrow from non-Hodgkin's diffuse lymphoma.

The most effective treatment for recurrent non-Hodgkin's lymphoma (NHL) appears to be a high-dose cytotoxic chemotherapy (HDC) followed by autologous bone marrow transplantation (ABMT). However, it has been suggested that the presence of occult lymphoma cells in harvested marrow may be responsible for a significant fraction of treatment failures after HDC/ABMT. The present study examined randomly accrued NHL patients, independent of their cytogenic grades, for the presence of cells bearing bcl-2/immunoglobulin heavy chain (IgH) gene rearrangements in lymph node (LN) biopsies and the bone marrow by polymerase chain reaction (PCR) and Southern blot hybridization combined with a classical culturing technique. Among 41 NHL patients examined, bcl-2/IgH translocations were evident in LN biopsies and marrow from each of 10 follicular lymphoma patients, but not in any samples from 31 newly diagnosed diffuse lymphoma patients. Marrow aspirates from several patients that were cultured using a one-week "triggering culture" followed by an extended period of conventional culture resulted in emergence of a monoclonal, IgH-rearranged, bcl-2-normal lymphoid cell population. Such outgrowth was specifically seen in cultures of diffuse lymphoma marrow (7 of 28 evaluable patients). Southern analysis for IgH rearrangement within LN biopsies and of cells cultured from marrow of individual diffuse lymphoma patients produced identical patterns, suggesting that the occult lymphoma cells present in harvested marrow were derived from the predominant lymphoma cell population represented within involved lymph nodes. The culture of histologically occult lymphoma from diagnostic marrow and analysis of the derived cells by Southern blot hybridization can be used to detect potentially aggressive lymphoma cells within harvested marrow, despite their lack of bcl-2 gene rearrangement.

Adult↗

Temporal bone histopathological and quantitative analysis of mitochondrial DNA in MELAS.

OBJECTIVES/HYPOTHESIS: Although hearing loss is common in MELAS (syndrome of mitochondrial encephalopathy, lactic acidosis and stroke-like episodes), the histopathology of the temporal bone has not been reported. The majority of cases of MELAS are linked to a mitochondrial DNA (mtDNA) mutation at nucleotide 3243. In MELAS, normal mtDNA and mutant mtDNA coexist in a heteroplasmic manner. The purpose of the study was to report the otopathological findings from two patients with MELAS and quantitative mtDNA analysis in the inner ear of one of these patients. STUDY DESIGN: Basic scientific histopathological examination and quantitative mtDNA analysis of the temporal bone. METHODS: Temporal bones were embedded in celloidin and sectioned for light microscopic study. Graphic reconstruction of the cochlea was performed using the method described by Schuknecht. For quantitative mtDNA analysis, total DNA from the membranous part of the inner ear was collected, amplified by polymerase chain reaction, and digested with the restriction enzyme. The percentage of mutant/total mtDNA was measured by the ratio of fluorescence intensity. RESULTS: Histopathological examination revealed severe degeneration of the stria vascularis and degenerative change of spiral ganglion cells in both patients. The quantitative DNA studies showed that the proportion of mutant to wild-type mtDNA was similar in both histologically affected and histologically unaffected tissues within the inner ear. CONCLUSION: Dysfunction of the stria vascularis and spiral ganglion cells causes sensorineural hearing loss in MELAS.

Adult↗

Effects of Nd:YAG and CO2 laser treatment and ultrasonic scaling on periodontal pockets of chronic periodontitis patients.

BACKGROUND: The aim of the present study was to compare the effectiveness of Nd:YAG and CO2 laser treatment to that of ultrasonic scaling used as monotherapies by examining clinical parameters, subgingival microflora, and interleukin-1 beta (IL-1beta) in gingival crevicular fluid (GCF). METHODS: Eighteen patients, each of whom had 2 or more sites with probing depth measuring > 5 mm, were included this clinical trial. The 41 sites were randomly assigned treatment with either Nd:YAG laser alone (n = 14, 100 mj, 20 pps, 2.0 W, 120 seconds), CO2 laser alone (n = 13, 2.0 W, 120 seconds), or ultrasonic scaling alone (n = 14, maximum power, 120 seconds). At baseline and at 1, 4, and 12 weeks, clinical measurements (plaque index, PI; gingival index, GI; probing depth, PD; clinical attachment level, CAL; and bleeding on probing, BOP) were performed and subgingival plaque and GCF sampled. A quantitative analysis of Porphyromonas gingivalis was carried out using real-time polymerase chain reaction (PCR) procedures. The amounts of IL-1beta were estimated by an enzyme-linked immunosorbent assay (ELISA). RESULTS: Decreased inflammation and PD were observed in all 3 groups after treatment. A microbiological analysis indicated significant decreases in P. gingivalis in the Nd:YAG and scaling groups at 1, 4, and 12 weeks compared to baseline (P < 0.05). The amount of GCF significantly decreased in the Nd:YAG and scaling groups at 12 weeks. The amount of IL-1beta increased in the CO2 group from baseline to 1 week (P < 0.05). The Nd:YAG group tended to show a decrease in IL-1beta from 1 to 12 weeks, although these data were not statistically significant. CONCLUSIONS: Our data suggest that Nd:YAG laser and ultrasonic scaling treatments showed significant improvements regarding the clinical parameters and subgingival microflora compared to the baseline, but no significant difference was observed between the 3 groups.

Analysis of Variance↗

Real-time PCR for quantification of Streptococcus mutans.

A real-time polymerase chain reaction (PCR) assay was developed for the quantification of Streptococcus mutans. Primers targeting gtf genes of S. mutans were designed and tested for their specificity using 28 oral streptococcal strains, three other bacterial strains, and human DNA. The primers could amplify specifically the target DNA fragment from a mixture of oral streptococcus genomic DNA containing about 10 fg to 10 ng of S. mutans genome DNA. The real-time PCR produced a linear quantitative detection range over concentrations spanning seven exponential values, with a detection limit of a few copies of S. mutans' genomic DNA per reaction tube. The results of the real-time PCR assay corresponded well to those of conventional culture assays for S. mutans in saliva samples. A real-time PCR assay for Streptococcus sobrinus and Streptococcus downei was also established and produced results that corresponded well to those from conventional culture assays for S. sobrinus in saliva samples. These assays will be useful as a new means to assess one of the important risk factors for caries.

Bacterial Typing Techniques↗

Insoluble powder formulation as an effective nasal drug delivery system.

PURPOSE: To evaluate the utility of insoluble powder formulation for nasal systemic drug delivery. METHODS: To compare the efficacy of liquid and powder formulations, the nasal absorption of drugs was examined in rats using hydrophilic compounds with various molecular weights (MW) such as phenol red, cyanocobalamin, and fluorescein isothiocyanate (FITC)-Dextrans, and several kinds of powder. Intranasal residence time was also compared among the different formulations. RESULTS: All the drugs examined were absorbed through the nasal mucosa to varying extent; their systemic bioavailability decreased with increasing MW. Insoluble calcium carbonate (CaCO3) powder formulation provided increased absorption of drugs over the wide range of MW from 354 to 77,000 Da. In the case of phenol red, intranasal administration as a CaCO3 powder formulation resulted in a plasma concentration profile similar to that of an intravenous bolus dose due to its very rapid and complete absorption from the nasal cavity. Furthermore, improved bioavailability of FITC-Dextran (MW 4,400; FD-4) was also achieved with other insoluble powders as well as CaCO3, but not with soluble powders such as lactose, d-sorbitol, and d-mannitol. Insoluble powder formulation prolonged the residence time of FD-4 within the nasal cavity. CONCLUSIONS: Insoluble powder formulations improve nasal bioavailability predominantly by retarding drug elimination from the absorption site and appear to be effective for nasal systemic drug delivery.

Administration, Intranasal↗

Ductal and acinar differentiation in pancreatic endocrine tumors.

Rare pancreatic endocrine tumors consisting of both exocrine and endocrine components have been reported sporadically. We investigated the ductal and acinar differentiation in pancreatic endocrine tumors. In immunohistochemical studies of 28 pancreatic endocrine tumors, staining with anti-carcinoembryonic antigen (CEA) or CA19-9 antibody indicated ductal differentiation, while staining with anti-amylase or anti-trypsin antibody indicated acinar differentiation. K-ras gene mutations and p53 gene alterations also were studied. Ten tumors were immunoreactive for CEA or CA19-9. Five tumors diffusely immunoreactive for CEA or CA19-9, in addition to endocrine markers, were diagnosed as duct-endocrine cell tumors of the pancreas. Two tumors diffusely immunoreactive for CEA or CA19-9 and also for pancreaticogut hormones as well as endocrine markers were diagnosed as duct-acinarendocrine cell tumors. These tumors showed uniform histologic features and synchronous ductal, acinar, and endocrine differentiation, distinct from the coexisting different cellular populations seen in collision tumors. All tumors were malignant. These duct-endocrine cell tumors or duct-acinar- endocrine cell tumors of the pancreas may be derived from a stem cell that retains the capability of expressing either an exocrine or endocrine phenotype. Only one K-ras gene mutation and no p53 gene alterations were detected in these tumors, which suggests that they constitute an entity with a different origin than ductal carcinomas.

Adult↗

[External quality assessment of the genetic testings for hematopoietic tumor, CML].

Genetic testings are commonly employed in various fields of clinical medicine and the test items performed at clinical laboratories are increasing rapidly in number. They are utilized to make early and/or definite diagnoses of infectious diseases, leukemia, cancers and molecular inherited diseases and also to monitor the progress of the diseases. However, these genetic testings except for infectious diseases have been developed independently at each clinical laboratory and the test results obtained at each laboratory are not always compatible each other. Under these situations it is widely expected to construct advanced genetic testing systems that can supply standardized data at any of domestic and international clinical laboratories. For the period from April, 1999 to March, 2002 three major clinical laboratories, SRL, Inc., BML, Inc. and MBC, Inc., were consigned by JBA (Japan Bioindustry Association) to collaborate in standardizing the evaluation methods for genetic testing systems among the clinical laboratories. The aim of the study is to develop the standardized genetic testing systems and to propose them as international standard operational procedures to the ISO/TC212 working group. Although one of the most important issues for standardization is the external quality assessment, they have not been carried out in reality. In this study we evaluated the difference of the genetic testing results obtained during the year of 2000 and 2001 among the clinical laboratories. The genetic testings for hematopoietic tumor, CML were selected to be evaluated since they are widely accepted as clinically useful tests.

Blotting, Southern↗

Detection of lymph node micrometastasis in esophageal carcinoma.

BACKGROUND/AIMS: The presence of lymph node metastasis is the most important prognostic factor in esophageal squamous cell carcinoma. Molecular biology techniques have improved the ability to recognize micrometastasis in lymph nodes, bone marrow, and peripheral blood. Previous studies have demonstrated that cytokeratin 19 reverse transcriptasepolymerase chain reaction can detect tumor cells even when lymph nodes appear normal histologically. However, the presence of pseudogenes for cytokeratin 19 have reduced the specificity of reverse transcriptase-polymerase chain reaction, thereby reducing its clinical worth as a sensitive diagnostic technique. METHODOLOGY: We examined the expression of mRNA for cytokeratin 19 using a newly designed set of primers, and compared the results with data from histologic examinations. Samples were obtained from tumors, intact tissues, resected lymph nodes and in 10 patients who underwent esophagectomy via right thoracotomy with lymph node dissection in the neck, mediastinum and abdomen. RESULTS: All tumors, non-cancerous tissues were positive for cytokeratin 19 by reverse transcriptasepolymerase chain reaction. However, 2 of the 6 lymph nodes that appeared normal on histologic examination were positive for cytokeratin 19; sensitivity and specificity were 100% and 67%, respectively. CONCLUSIONS: Reverse transcriptase-polymerase chain reaction using new primers for cytokeratin 19 detected micrometastasis in specimens of lymph nodes from patients with squamous cell carcinoma. This method may increase the accuracy of tumor staging and provide clinicians with valuable information that will help individualize treatment options.

Esophageal Neoplasms↗