Prognostic impact of PCR-based identification of isolated tumour cells in the peritoneal lavage fluid of gastric cancer patients who underwent a curative R0 resection.

Identification of cancer cells in the peritoneal cavity could influence therapy and outcome of gastric carcinoma patients. The objective of this study was to evaluate the clinical impact of the real-time quantitative polymerase chain reaction-(PCR) based identification of isolated tumour cells in the peritoneal lavage fluid of gastric carcinoma. The peritoneal lavage fluid of 116 patients with gastric cancer was sampled at laparotomy. After RNA extraction and reverse transcription, real-time quantitative PCR was performed using the primers and probes for carcinoembryonic antigen (CEA) and cytokeratin-20 (CK20). When either the CEA mRNA or CK20 mRNA level of the sample was over the cutoff value, the sample was determined to be PCR-positive. Forty-six (40%) of the 116 patients were PCR-positive and 30 (65%) of the 46 PCR-positive patients died as a result of recurrent peritoneal dissemination. The prognosis of the 46 PCR-positive patients was significantly (P<0.001) worse than that of 70 PCR-negative patients. Furthermore, in 80 of the cases with a curative R0 resection, 15 of the patients with PCR-positive findings had a significantly (P<0.001) poorer prognosis than the 65 PCR-negative patients. The prognosis of the PCR-positive patients was significantly poorer than that of the PCR-negative patients in the T3 (P<0.0001) and T4 (P=0.048) subgroups. In a multivariate analysis of the 80 cases with a curative R0 resection, the real-time quantitative RT-PCR (CEA and/or CK20) levels indicated that they were independent prognostic factors. The real-time quantitative RT-PCR analysis of the CEA and/or CK20 transcripts in the peritoneal lavage fluid is useful for predicting the peritoneal recurrence in patients who are undergoing a curative resection for gastric cancer.

Efficacy of Helicobacter pylori eradication on the chronic mucosal inflammation of the remnant stomach after distal gastrectomy for early gastric cancer.

Although eradication of Helicobacter pylori (Hp) after early gastric carcinoma has been recommended, very limited studies have been reported and the method differs from standard therapy. Here, we attempted the eradication of Hp in the remnant stomach after surgery for primary gastric cancer with the standardized method. We examined efficacy and the safeness of the treatment. Thirty-three H. pylori-positive patients after distal gastrectomy were treated with proton pump inhibitor (PPI)-based triple therapies. After eradication, endoscopic and histological changes were classified on the basis of the Updated Sydney System. The eradication rate in the remnant stomach was 90.9% (30 out of 33 cases) after triple therapy. Temporal minor side effects were notified in 3 cases. After eradication, the remnant stomach showed significant decreases in inflammation- and activity-scores. Moreover, significant improvement in glandular atrophy to normal mucosa was found. In conclusion, PPI-based standard therapy is just as effective for Hp eradication in the remnant stomach than it is in the non-operative stomach. Eradication therapy could be performed safely and resulted in a significant improvement in inflammation and atrophy of the mucosal layer in the remnant stomach after early gastric cancer surgery.

Isolation of peptides useful for differential diagnosis of Crohn's disease and ulcerative colitis.

BACKGROUND: Phage displayed random peptide technology has been utilised to identify binding epitopes of antibodies or receptor ligands. AIM: To isolates peptides from a phage library which are specifically recognised by antibodies in serum from patients with Crohn's disease (CD). METHODS: A phage displayed random peptide library composed of nine amino acids was established and sequentially screened using serum immunogloblin G obtained from CD patients. RESULTS: Five different CD specific peptides were isolated from the phage library. No homology in amino acid sequences was observed among four (CDP-1, -3 to -5) of the five peptides exhibiting different binding characteristics with each CD patient's serum. In contrast, two peptides (CDP-1 and -2) had similar amino acid sequences and similar binding characteristics. Four multiple antigenic peptides (MAP, CDP-1, -3 to -5) were synthesised, and an enzyme linked immunosorbent assay (ELISA) using the four peptides was developed to detect serum antibodies against them. Fifty two of 92 CD patients (56.5%) were detected by ELISA, none of 20 ulcerative colitis (UC) patients, only one of 25 duodenal ulcer patients, and only three of 48 healthy subjects. CONCLUSIONS: ELISA using the four peptides isolated in this study may be useful for the differential diagnosis of CD and UC.

Differentiated basal cell carcinoma in a Cape clawless otter (Aonyx capensis).

Basal cell carcinoma with folliculoapocrine differentiation was diagnosed in a male Cape clawless otter (Aonyx capensis) aged >8 years. A tumour mass in the left submandibular region was removed surgically, but another tumour subsequently appeared on the left cheek. In addition, necropsy revealed a subcutaneous tumour mass at the excision site. Histologically, the tumours consisted of lobules or islands of basaloid cells, frequently with central keratinization and tubular structures. The presence of isthmic keratinization and apocrine differentiation was confirmed by immunolabelling for cytokeratins, and a few cytokeratin-positive tumour cells were found in a submandibular lymph node. The neoplasm, characterized by its metachronous and recurrent nature, metastasis to the local lymph node and amyloidosis, closely resembled human differentiated basal cell carcinoma, both clinically and pathologically.

Combined use of adenosine and amrinone inhibits reperfusion injury of rat liver.

Although intraportal infusion of adenosine suppressed the oxidative stress caused by activated neutrophils and attenuated ischemia-reperfusion injury of canine liver, high doses of adenosine elicit systemic hypotension. The present work demonstrates that combined use of low doses of adenosine and amrinone, a phosphodiesterase inhibitor, strongly inhibited reperfusion injury of the liver without eliciting hypotension. After 45 min ischemia followed by 60 min reperfusion of rat liver, low doses of adenosine and amrinone were administrated intraportally, resulting in significantly increased hepatic levels of cGMP, cAMP, nitrite plus nitrate in plasma, and decreased alanine aminotransferase in plasma without changing hemodynamics. Thus, intraportal administration of low doses of adenosine and amrinone increased the cyclic nucleotides, thereby improved microcirculation and attenuated reperfusion injury of the liver.

Experimental transmission of sheep-associated malignant catarrhal fever from sheep to Japanese deer (Cervus nippon) and cattle.

The assumption that sheep carry ovine herpesvirus-2 (OvHV-2), the causative agent of sheep-associated malignant catarrhal fever (SA-MCF), is widely accepted, albeit OvHV-2 has not been isolated. We attempted experimental contact transmission of MCF from Japanese sheep persistently infected with OvHV-2 to Japanese deer (Cervus nippon) and cattle. In Experiment 1, a deer was kept in close quarters with an infected ewe. In Experiment 2, a second deer was kept with the same ewe. In Experiment 3, two cows were each kept with two infected wethers. In Experiment 1, the deer developed clinical signs at 138 days after first contact and then died. OvHV-2 genes by polymerase chain reaction (PCR) and fluorescent antibodies to Alcelaphine herpesvirus-1 were detected in the affected deer. Moreover, sequences of PCR products (423bp), obtained by amplification of materials from the sheep and from the affected deer, coincided. These results clearly confirmed that the sheep was a carrier of OvHV-2, and that this virus had induced SA-MCF in a deer. In other experiments, no OvHV-2 infection occurred in deer and cattle during the 6-18 months periods of contact, though viral genes were detected in the nasal swabs and white blood cells of the sheep. To our knowledge, this is the first report on successful experimental transmission of MCF from OvHV-2-infected sheep to deer.

Granular cell tumor of the male breast: report of a case.

We treated a 35-year-old male with a granular cell tumor in the right breast. Physical examination revealed a solid, flattened, round 3.2 x 2.5-cm mass with an irregular surface, covering skin fixation and right axillary lymphadenopathy. Mammography revealed a well-demarcated high-density mass with a minimal starburst appearance. Ultrasonography revealed a hypoechoic, nonhomogeneous mass with an acoustic shadow. Several enlarged lymph nodes in the right axilla were removed at the time of breast tumor excision. Histologically, the tumor featured nests of round or polygonal cells with abundant eosinophilic cytoplasmic granules and small round nuclei, and the enlarged lymph nodes in the right axilla exhibited no metastasis. Immunohistochemically, there was positive staining for S-100 protein, neuron-specific enolase, and vimentin. The tumor also stained for macrophage CD-68, alpha1-antichymotrypsin, and myoglobin. These immunohistochemical findings suggested the tumor cells to be undifferentiated mesenchymal cells which demonstrated the properties of neurogenic cells and histiocytes.

Highly sensitive urine-based enzyme-linked immunosorbent assay for detection of antibody to Helicobacter pylori.

BACKGROUND: Enzyme-linked immunosorbent assay (ELISA) has been widely used for detection of Helicobacter pylori (H. pylori), but sample collection is often invasive, complicated, and expensive. Urine samples can be obtained noninvasively and are easier and safer to handle than serum samples. A urine-based ELISA, if found to be accurate, would therefore be a useful alternative to serum-based tests for H. pylori. METHODS: An ELISA method was developed for detection of antibodies to H. pylori in urine. Its sensitivity and specificity were compared with those of three commercially available serum-based ELISA kits and the 13C urea breath test (13C-UBT) using samples from 99 healthy volunteers and 20 patients with gastric disorders. RESULTS: With the assumption that 13C-UBT results are 100% accurate, the sensitivity and specificity of the urinary ELISA were 99% and 100%, respectively, and the accuracy (99%) was superior to those of the three serum ELISAs tested. Immunostaining profiles on Western blot analysis using serum samples were almost identical to those obtained using paired urine samples. CONCLUSIONS: These findings suggest that the differences observed among ELISA test results may be due principally to differences between the profiles of antigen coated on plates for the assays, rather than to differences between antibodies in serum and urine. The urine-based ELISA (URINELISA H. pylori) developed in this study is very accurate and would be useful for screening H. pylori infection as an alternative to serum ELISAs.

Degradation of Derivatives of N-Acetyl-D-glucosamine by Rhodococcus rhodochrous IFO 15564: Substrate Specificity and Its Application to the Synthesis of Allyl α-N-Acetyl-D-glucosaminide.

The substrate specificity was studied for the metabolic degradation of N-acetyl-D-glucosamine (GlcNAc) derivatives by Rhodococcus rhodochrous IFO 15564 which possesses N-acetyl-D-glucosamine deacetylase as a key-step enzyme. This microorganism degraded a wide range of substrates with modified N-acyl groups. The metabolizing activity of this strain became low to the substrates substituted at 1,3,4,6-positions of GlcNAc, and GlcNAc itself was suggested to be metabolized via an open-chain aldehyde form. Based on these results, a simplified procedure for the isolation of allyl α-N-acetyl-D-glucosaminide from an α, ß-anomeric mixture was developed by selectively hydrolyzing the ß-anomer with Jackbean ß-N-acetyl-D-glucosaminidase and subsequently degrading the resulting N-acetyl-D-glucosamine in the reaction mixture with this microorganism.

Method for detection of epsilon-secondary structure in the precore region of human hepatitis B virus DNA using a fluorescence-based polymerase chain reaction-single-strand-conformation polymorphism technique with capillary electrophoresis.

A portion of the precore region of the human hepatitis B virus (HBV) genome is the signal sequence with an epsilon secondary structure, which plays a role in the encapsidation of HBV pregenome RNA. To determine the genetic mutations which occur in the precore region of HBV, we have devised a typing method using a fluorescence-based polymerase-chain-reaction-single-strand conformation polymorphism technique with automated capillary electrophoresis (CE-FSSCP). Using the cloning sequencing method, we analyzed serum samples from 10 patients with hepatitis B, and detected three types of HBV-DNA including two mutants which are crucial to the function of the encapsidation sequence: position 1896 G (guanine) to A (adenine, stop codon), position 1899 G to A, and wild-type. We performed CE-FSSCP analysis of these three types of HBV-DNA and described conditions for determination of the mutations which play roles in the encapsidation of the HBV pregenome. The two types of epsilon mutants and wild-type DNA were identified as separate individual peaks respectively. The observed migration times of the three types of DNAs agreed fairly well with estimates obtained from total RNA secondary structure energy.

Fluorescence-based polymerase chain reaction-single-strand conformation polymorphism analysis of p53 gene by capillary electrophoresis.

Mutation of the p53 gene plays an important role in neoplastic progression in human tumorigenesis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) techniques are now available for the detection of point mutations. The original method using polyacrylamide gel electrophoresis is disadvantageous, particularly for clinical tests and for analysis of large numbers of samples. Therefore, using an automated capillary electrophoresis (CE) technique with a molecular-sieving polymer solution, we have devised a completely automatic fluorescence-based PCR-SSCP system (CE-FSSCP) for the differential detection of point mutations that dose not require SSCP with radioisotopes and polyacrylamide gels. The automatic CE-FSSCP system was developed for reproducible operations in the denaturation of double-stranded DNA and electrophoresis of single-stranded DNA. The detection system consists of a 100 W I2 lamp and photomultiplier. We performed CE-FSSCP with a 2% linear polyacrylamide polymer solution containing 5% glycerol. Four tissue specimens of lung tumors with mutations in exon 7 of the p53 gene were found to have mutant alleles; six-base-pair deletion at codons 247-248, a one-base-pair deletion at codon 260, a one-base-pair deletion at codon 244 and a GGC to CGC substitution at codon 244. We expect this technique to prove useful for the clinical DNA diagnosis of human cancers, determination of the therapeutic effect of anticancer agents and for the study of the molecular aspects of the mechanisms involved in the pathogenesis of human cancers.

Molecular basis of leukocyte adhesion molecules in early-onset periodontitis patients with decreased CD11/CD18 expression on leukocytes.

We analyzed the cell-cell adherence related to CD11/CD18 and CD18 mRNA in individuals with decreased CD11/CD18 expression on their neutrophil surface. Epstein Barr virus-transformed B cell lines were developed from one localized juvenile periodontitis (LJP) patient with decreased CD11/CD18 in the peripheral blood neutrophils and without systemic diseases; two siblings with generalized prepubertal periodontitis (GPP) caused by leukocyte adhesion deficiency (LAD); another LJP patient; one localized prepubertal periodontitis (LPP) patient; and two healthy subjects. Adhesion of leukocytes to each other was measured as cluster formation by aggregation assay. The length and the amount of CD18 mRNA expressed in the cell lines were analyzed by Northern blotting using the 32P-labeled CD18 cDNA. The coding region of the mRNA was analyzed by the reverse transcription-polymerase chain reaction method. Base-mismatches between CD18 mRNA and the 32P-labeled RNA probe synthesized from CD18 cDNA were analyzed by RNase protection assay. In the adherence assay, cells from the LJP patients with decreased CD11/CD18 formed more clusters of smaller size and fewer cells than those of the other subjects. The cells from GPP and LAD patients did not aggregate and did not form clusters either in the absence or presence of PMA. There were no differences in the length and the amount of mRNA between the LJP patients and the other subjects, while GPP-LAD patients expressed a small amount of long mRNA. The whole coding region (2,313 base pairs) of all subjects was amplified except for the GPP-LAD patients, and the 5'-region (1,119 base pairs) was amplified from all subjects.(ABSTRACT TRUNCATED AT 250 WORDS)

DNA typing of HLA-B gene in Takayasu's arteritis.

Sixty-four patients with Takayasu's arteritis and 156 healthy individuals in the Japanese population were examined for HLA-B specificity at the DNA level by DNA typing using polymerase chain reaction (PCR)/sequence-specific oligonucleotide probe (SSOP) analysis and by subsequent sequencing analysis. The frequency of epitope combination group-B52 (EC-B52) corresponding precisely to HLA-B52 specificity and that of EC-B39.2 which is a newly-identified subtype of HLA-B39 specificity were increased in the patient group. These two disease-associated HLA-B alleles share an epitope composed of 63Glu and 67Ser. Because two HLA-B alleles, HLA-B51 and B39.1, which are similar but different at the epitope from HLA-B52 and B39.2, respectively, are not associated with Takayasu arteritis, 63Glu and 67Ser are supposed to be involved in the pathogenesis.

ras mutations in endocrine tumors: mutation detection by polymerase chain reaction-single strand conformation polymorphism.

To elucidate the molecular basis for endocrine tumorigenesis, ras mutations in human endocrine tumors were analyzed using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Mutations of the H-, K-, N-ras genes were examined in genomic DNAs from 169 successfully amplified primary endocrine tumors out of 189 samples. Four out of 24 thyroid follicular adenomas analyzed contained mutated N-ras codon 61, and one contained the mutated H-ras codon 61. One of the 19 pheochromocytomas revealed mutation of the H-ras codon 13. No mutations of the ras gene were detected in pituitary adenomas, parathyroid tumors, thyroid cancers, endocrine pancreatic tumors, and adrenocortical tumors. Based on these findings we conclude that activation of the ras gene may play a role in the tumorigenesis of a limited number of thyroid follicular adenomas and pheochromocytomas, and that mutation of the ras gene is not frequent in other human endocrine tumors.

Molecular cloning and characterization of the fusaric acid-resistance gene from Pseudomonas cepacia.

Fusaric acid-resistance genes (fus) were isolated from Pseudomonas cepacia. The nucleotides of the 5437 base pairs containing the fus genes were sequenced.