Non‐Helicobacter pylori helicobacters (NHPHs) besides H. pylori infect human stomachs and cause chronic gastritis and mucosa‐associated lymphoid tissue lymphoma. Cholesteryl‐α‐glucosides have been identified as unique glycolipids present in H. pylori and some Helicobacter species. Cholesterol‐α‐glucosyltransferase (αCgT), a key enzyme for the biosynthesis of cholesteryl‐α‐glucosides, plays crucial roles in the pathogenicity of H. pylori. Therefore, it is important to examine αCgTs of NHPHs.

A spiral bacterium(SH9),morphologically different from Helicobacter pylori(H.pylori),was found in a 62-yearold woman’s gastric mucosa.Gastroscopic examination revealed multiple gastric ulcers near the pyloric ring;mapping gastric biopsy showed mild mononuclear infiltration with large lymphoid follicles in the antrum,without corpus atrophy.Urea breath test and H.pylori culture were negative,but Giemsa staining of biopsies revealed tightly coiled bacteria that immunostained with anti-H.pylori antibody.Sequencing of SH9 16S rRNA and the partial urease A and B subunit genes showed that the former sequence had highest similarity(99%;1302/1315 bp)to Helicobacter heilmannii(H.heilmannii)sensu stricto(H.heilmannii s.s.)BC1 obtained from a bobcat,while the latter sequence confirmed highest similarity(98.3%;1467/1493 bp)to H.heilmannii s.s.HU2 obtained from a human.The patient was diagnosed with multiple gastric ulcers associated with H.heilmannii s.s.infection.After triple therapy(amoxicillin,clarithromycin,and lansoprazole)with regimen for eradicating H.pylori,gastroscopy showed ulcer improvement and no H.heilmannii s.s.upon biopsy.

Background: To study the functions of residues gamma 326Cys and gamma 339Cys in the assembly and/or secretion of fibrinogen, recombinant fibrinogens were synthesized to replicate naturally occurring gamma 326Tyr and gamma 326Ser variants, along with gamma 326Ala and gamma 339Ala variants.Methods: A fibrinogen gamma-chain expression vector was altered and transfected into Chinese hamster ovary (CHO) cells. Cell lysates and culture media of the established cell lines were subjected to ELISA and immunoblotting analysis. In addition, pulse-chase analysis was performed.Results: The CHO cells synthesized mutant gamma-chains and assembled these into fibrinogen in the cells, although these variant fibrinogens were barely secreted into the culture media. Pulse-chase analysis indicated that the rates of both assembly and secretion of the variant fibrinogens were lower than that of normal fibrinogen.Conclusions: The present study indicated that the 326-339 intrachain disulfide bond has a crucial role in maintaining the tertiary structure of the C-terminal domain of the gamma-module, which is necessary for fibrinogen assembly and specifically secretion. A combination of the present results and observations from naturally occurring heterozygous cases of gamma 326Tyr and gamma 326Ser suggest that heterozygous fibrinogen molecules containing variant gamma-chains might be secreted into plasma and show impaired fibrin polymerization, resulting in a phenotype of hypodysfibrinogenemia. (c) 2009 Elsevier Ltd. All rights reserved.

Cutaneous Epstein‐Barr virus (EBV)‐associated B‐cell lymphoma (EBVBL) in non‐immunocompromised patients is very rare. Here, we report a case of cutaneous EBVBL in a 72‐year‐old Japanese woman without any signs of immunosuppression. She showed repeated high fever and skin eruptions on the face, limbs and palms. Histological diagnosis was diffuse large B‐cell lymphoma. EBV infection was detected by in situ hybridization and Southern blotting. Immunostaining for viral proteins showed the patient to be positive for latent membrane protein 1 (LMP‐1) and negative for Epstein‐Barr virus nuclear antigen‐1 (EBNA‐2), indicating that a type II latency EBV infection pattern.

Chimerism analysis by polymerase chain reaction amplification of short tandem repeats (PCR-STR) has become a routine diagnostic procedure for evaluating grafts and assessing the likeliness of original disease recurrence after allogeneic stem cell transplantation. Following a sex-mismatched hematopoietic stem cell transplantation (HSCT), we monitored the clinical course of a 61-year old male AML M6 patient with trisomy 8 using PCR-STR with a TH01 locus on 11p15 and fluorescence in situ hybridization (FISH) analysis specific for alpha satellite DNA on chromosome 8. Ten months after HSCT, FISH analysis showed 24.8% recipient cells, but PCR-STR demonstrated 100% donor type chimerism. Further XY FISH analysis of May-Grünwald-Giemsa-stained bone marrow samples clearly demonstrated relapse of the original disease and G-banding analysis of bone marrow samples at relapse showed that an additional chromosomal abnormality, del(11) (p10), had deleted the PCR-STR detection site in all recipient type cells. As such, clinicians should consider the possibility that unexpected karyotype changes may invalidate PCR-STR analysis findings, especially when conflicting results appear among chimerism analyses.

Objective: Human serum sphingomyelin (SM) and phosphatidylcholine (PC) play important roles in the development of atherosclerosis. However, there are no rapid and sensitive methods for SM and PC measurement. The present report describes a novel enzymatic method for measuring SM, PC and lysophosphatidylcholine (lyso-PC) levels in plasma and lipid extracts.Design and methods: The total choline-containing phospholipids (total PL), SM and PC were measured using a two-reagent system involving specific enzymes for choline-based phospholipids. The procedure was performed using either microplate or automatic analyzer technology. The concentration of lyso-PC was calculated by subtracting the concentration of SM plus PC from the total PL concentration.Results: Assay results showed linear correlations between sample concentration and absorbance. The within-run and between-run coefficients of variation for PC, SM, and lyso-PC concentrations were 2.0-4.4% for the microplate analyzer and 0.9-2.9% for the automatic analyzer. Analysis of normal human serum showed that the total PL concentration strongly correlated with the SM plus PC concentration (r=0.9850). There were moderate correlations between serum PC and SM levels (r=0.6228) and between serum PC and lyso-PC levels (r=0.7806). SM, PC, and lyso-PC levels in normal human serum (n=50) were 0.54 +/- 0.07, 1.99 +/- 0.22 and 0.60 +/- 0.15 mmol/L, respectively.Conclusion: The present enzymatic method allowed for rapid, simple, and accurate measurement of SM, PC, and lyso-PC levels in lipid extracts and in serum. The method is suitable for both microplate and automatic analyzer assays. (C) 2008 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.

We evaluated three test kits for fibrin degradation products (FDP) D-dimer. We found that six of 217 plasma sample values obtained by Nanopia test were markedly higher than the values obtained using the other two kits. The regression equation for 211 samples (excluding six) was y=0.64x+3.05 (y: Nanopia, x: LIAS AUTO) and the correlation coefficient was 0.915. Therefore, we classified these samples into three categories, namely correlated(y< 1.0x), incompatible (y= 1.0x-2.9x) and markedly incompatible (y> or =3.0x). Selected samples, eight correlated, four incompatible and four markedly incompatible, were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by Western blotting(WB). WB analysis using anti-fibrinogen antibody showed that both high molecular weight fragments of cross-linked fibrin (HMW-XDP) and DD/E fragments were present in the correlated samples, but there was less HMW-XDP than DD/E in the incompatible samples and mostly DD/E (HMW-XDP was significantly less than DD/E) in the markedly incompatible samples. These data suggest that plasma FDP samples that contain mostly DD/E and little HMW-XDP demonstrated markedly incompatible values using the three D-dimer test kits. These data was reflected by markedly elevated plasmin alpha2-plasmin inhibitor complex values in the incompatible and markedly incompatible samples. Unfortunately, we did not directly demonstrate these phenomena by WB analysis with two anti-D-dimer antibodies used Nanopia or LPIA reagent. In the near future, we expect that standardization of FDP D-dimer assay will be accomplished.

Surveillance of Helicobacter pylori antimicrobial susceptibility reflecting the general population in Japan is limited. The antimicrobial susceptibilities of 3,707 H. pylori strains isolated from gastric mucosa samples of previously untreated patients diagnosed with gastroduodenal diseases at 36 medical facilities located throughout Japan between October 2002 and September 2005 were evaluated. Using an agar dilution method for antimicrobial susceptibility testing of H. pylori, the MIC distributions and trends during the study period for clarithromycin, amoxicillin, and metronidazole were studied. While the MIC (50) and MIC90 for clarithromycin did not change during the 3-year period, the MIC80 showed a 128-fold increase. Furthermore, the rate of resistance increased yearly from 18.9% (2002 to 2003) to 21.1% (2003 to 2004) and 27.7% (2004 to 2005). With a resistance rate of 19.2% among males compared to 27.0% among females, a significant gender difference was observed (P < 0.0001). Our study shows that in Japan, there is an evolving trend towards increased resistance to clarithromycin with geographical and gender differences as well as between clinical disease conditions. No significant changes in resistance were observed for amoxicillin and metronidazole during the period. While the benefit of H. pylori antimicrobial susceptibility testing has been debated in Japan, current empirical regimens are not based on susceptibility data representative of the general population. The development of an effective H. pylori eradication regimen in Japan will require continued resistance surveillance as well as a better understanding of the epidemiology of resistance.

Esophageal gland duct adenomas are extremely rare tumors. Here, we report the case of a 75-year-old Japanese man who had undergone total gastrectomy for advanced gastric cancer. Esophageal gland duct adenoma was incidentally found in the lower esophagus. It appeared to be detached from the site of gastric cancer and was well demarcated without a capsule. Histologic analysis revealed papillary and cystic structures mainly comprising eosinophilic cells with minimum nuclear atypia. Immunohistochemical analysis revealed that the tumor were diffusely positive for the S100 protein with preserved alpha-SMA-positive myoepithelial cell layers and a characteristic cytokeratin expression pattern similar to that in normal esophageal gland ducts (CK5/6+++, CK7+++, CK17+, CK18+, CK19+++, CK20-, HMWCK+++). In addition, differentiation into the terminal duct was confirmed by a combination of mucin staining and immunohistochemical and ultrastructural examinations. This is the first report that refers to the ultrastructural findings of an esophageal gland duct adenoma and describes terminal duct differentiation. We believe that the possibility of an esophageal gland duct adenoma should be considered when diagnosing a ductal or glandular lesion of the esophagus.

We previously reported a case of heterozygous β-thalassaemia with IVS1-1G > C substitution in the β-globin gene and a non-detectable level of mutant mRNA in the patient's reticulocytes. The purpose of this study was to determine whether the transcription and RNA splicing and processing of the mutant gene occurred. We analysed the expression of the mRNA encoded by the cloned mutant gene in COS-1 cells by reverse transcription-polymerase chain reaction followed by agarose gel electrophoresis and nucleotide sequencing. The G > C mutation completely inactivated the normal 5- splice site and resulted in the activation of two cryptic 5- splice sites, located 16 and 38 nt upstream of the normal site. The usage of these two cryptic sites accords with the findings of reports on IVS1-1G > A or IVS1-1G > C substitution of exon 1 of the β-globin gene. Additional experiments that involved transfection of equal amounts of both normal and mutant vectors into COS-1 cells indicated the presence of mutant mRNAs. In conclusion, the β-thalassaemia gene (IVS1-1G > C) was expressed in transfected cells, but showed aberrant RNA splicing. Further studies will be required to clarify the molecular mechanism that results in severe reduction in the mutant mRNA level in vivo.

Podoplanin, which is immunoreactive to D2-40 antibody, is reportedly expressed in lymphatic vessels in non-neoplastic tissues, and also in vascular and non-vascular tumors. However, its expression in non-neoplastic and neoplastic liver tissues has not been well documented. In this study, we examined podoplanin expression in specimens from 10 normal livers and 73 cases of liver tumors: hemangioma (16 cases), epithelioid hemangioendothelioma (9 cases), angiosarcoma (4 cases), angiomyolipoma (7 cases), hepatocellular carcinoma (11 cases), intrahepatic cholangiocarcinoma (11 cases), and metastatic liver cancer (15 cases). We compared levels of podoplanin and other endothelial markers (CD31, CD34, and factor VIII) in liver tumors. In the normal liver, podoplanin was expressed in lymphatic endothelium, nerve fibers, and mesothelium in the hepatic capsule, but not observed in any cells within hepatic lobules. Among liver tumors, podoplanin was specifically expressed in seven of nine cases (78%) of epithelioid hemangioendothelioma but not in other hepatic tumors. The expression of CD31, CD34, and factor VIII was observed in endothelial cells in all cases of hemangioma, epithelioid hemangioendothelioma, angiosarcoma, and angiomyolipoma with one exception, a case of epithelioid hemangioendothelioma which was without CD31 expression. Interestingly, the intensity of podoplanin expression was negatively correlated with the expression of CD34 and factor VIII. In conclusion, podoplanin would be useful as a diagnostic marker for epithelioid hemangioendothelioma in liver tumors.

This study used matrix-assisted laser desorption and ionization time-of-flight mass spectrometry (MALDI-TOF MS) to identify all lipid classes in human serum lipoproteins. After the major lipoproteins classes were isolated from serum by ultracentrifugation, the lipids were extracted and mixed with 2,5-dihydroxybenzoic acid (2,5-DHB) dissolved in Folch's solution (chloroform/methanol 2:1, v/v). MALDI-TOF MS analysis of the samples identified phospholipids (PLs), lysophospholipids (lysoPLs), sphingolipids (SLs), triglycerides (TGs), cholesteryl esters (CEs), and free cholesterol; it also showed the characteristics of individual fatty acid chains in serum lipids. MALDI-TOF MS allowed analysis of strongly hydrophobic and non-polar molecules such as CEs and TGs as well as hydrophilic molecules such as phospholipids. Direct analysis of fatty acids was not possible. The concentrations of lipids were not consistent with the ion peak intensities, since the extent of polarity affected the ionization characteristics of the molecules. However, lipid molecules with similar molecular structures but various fatty acid chains, such as phosphatidylcholine (PCs), were analyzed quantitatively by MALDI-TOF MS. Quantitative measurement of cholesterol was possible with the use of an internal standard. This study shows that MALDI-TOF MS can be used for direct investigation and quantitative analysis of the phospholipid composition of serum lipoproteins.

We analyzed the clinical factors resulting in hypofibrinogenemia, which is defined as less than 100mg/dl of plasma fibrinogen values determined by a procedure based on the Thrombin-time method. Within a 12-month period, we assayed 5,746 patients (19,309 plasmas) and found 113 patients (1.97%) with hypofibrinogenemia. We categorized these patients as having decreased synthesis of fibrinogen (less than 3.0g/dl of albumin, 140 IU/l of Cholinesterase, and/or 50% on Hepaplastin Test), increased consumption of fibrinogen (more than 10 microg/ml of FDP D-dimer), known side effect of L-asparaginase administration, or other causes. Details are follows: 1) decreased synthesis: 26 patients, suspected of decreased synthesis (albumin: 3.1-3.4 g/dl): 4 patients, 2) increased consumption: 15 patients, suspected of increased consumption (FDP D-dimer: 5.0-9.9 g/dl): 1 case, 3) decreased synthesis combined with increased consumption: 24 patients, suspected of decreased synthesis and/or suspected of increased consumption: 14 patients, 4) side-effect of L-asparaginase administration: 24 patients, 5) heterozygous dysfibrinogenemia: 1 patient, 6) heterozygous fibrinogen deficiency: 1 patient, suspected of heterozygous fibrinogen deficiency: 1 patient, 7) unidentified: 2 patients with West syndrome treated with a combination of ACTH and valproic acid. Three patients with dysfibrinogenemia or fibrinogen deficiency showed normal or slightly prolonged PT values and normal APTT values. These data and our previous reports suggest that heterozygous patients with dysfibrinogenemia or fibrinogen deficiency do not demonstrate markedly prolonged PT and APTT values, differing from patients with afibrinogenemia.

Distinguishing cutaneous metastasis of gastric cancer from primary sweat gland carcinoma can be problematic in some cases, especially with a single lesion. Previously we showed that a monoclonal antibody HIK1083 directed to α1,4-GlcNAc-capped O-glycans expressed in gastric gland mucin reacts to gastric cancer cells. By contrast, it was reported that immunohistochemistry for cytokeratin 20 (CK20) may be helpful in the differential diagnosis between cutaneous metastasis of gastric cancer and primary sweat gland carcinoma. Here, we immunohistochemically examined the expression of α1,4-GlcNAc-capped O-glycans and CK20 in 7 primary sweat gland carcinomas, 7 cutaneous metastases of gastric cancer, and 21 cutaneous metastases of other origin including breast, lung, colorectum, prostate, thyroid and pancreas using HIK1083 and CK20-specific Ks 20.8 antibodies and then assessed the usefulness of these antibodies in distinguishing cutaneous metastases of gastric cancer from primary sweat gland carcinoma and other cutaneous metastatic tumors. Both α1,4-GlcNAc-capped O-glycans and CK20 were positive in 5 of 7 cases of cutaneous metastases of gastric cancer, while neither α1,4-GlcNAc-capped O-glycans nor CK20 were detected in any of the primary sweat gland carcinomas. By contrast, α1,4-GlcNAc-capped O-glycans was not detected in any of the cutaneous metastases other than that of gastric cancer, whereas CK20 was detected in cutaneous metastases of colorectal cancer (2/2), breast cancer (2/13), and lung adenocarcinoma (1/3). These findings indicate that immunohistochemistry using HIK1083 antibody is superior to immunohistochemistry for CK20 in distinguishing cutaneous metastasis of gastric cancer from primary sweat gland carcinomas and other cutaneous metastases.

Clinical laboratories need to know and manage the costs of laboratory tests, because they need financial data (1) to estimate costs per patient, (2) to request a budget to buy equipment, and (3) to improve their work; however, less than 40% laboratories practice cost management. In 2002, Shinshu University Hospital began to assess the costs of laboratory tests, but it was difficult to evaluate the quality of our cost management because there are few data and papers about the costs of laboratory tests in Japan. In this article, we practiced cost analysis using Shinshu University Hospital's data for 3 years (2002-2004), and studied the features of laboratory test costs and the problems of laboratory cost management. As a result, we listed 7 points to check cost management in clinical laboratories. This check list was established using only one data from our hospital. So, we suggest the benchmarking laboratory test costs between laboratories of the same type of hospitals or various laboratories.

Biliary cystic tumors, which are also called biliary cystadenoma and cystadenocarcinoma, are thought to be a heterogeneous disease entity, and some of them are known to show a luminal communication to the bile duct. In this study, we examined the clinicopathological features of nine cases of biliary cystic tumors with bile duct communication. They were composed of five males and four females with an average age of 67 years (52–84 years). They were multilocular (eight cases) or unilocular (one case), and all cases contained mucinous fluid. A direct luminal communication with the bile ducts was identified in five cases on preoperative or intraoperative cholangiographies. Biliary cystic tumors examined in this study were histologically adenoma (one case), adenocarcinoma in situ (six cases), and adenocarcinoma associated with microinvasive mucinous carcinoma (two cases). One case of adenocarcinoma in situ also had the adenoma component (adenocarcinoma in adenoma). Dysplastic mucinous epithelium proliferated in flat, micropapillary and papillary fashions within the intracystic spaces. Intraepithelial neoplasm was observed within non-dilated adjacent bile ducts, suggesting a direct luminal communication between the cystic tumors and the bile duct. Ovarian-like stroma was not observed in their walls in any cases. Immunohistochemically, seven cases expressed MUC1 or MUC2 in the neoplastic biliary epithelium. All cases except one were alive without any evidences of tumor recurrence after total excision (3–156 months after surgery). These clinicopathological features resembled those of intraductal papillary neoplasm of the bile duct, which had been reported as a biliary counterpart of pancreatic intraductal papillary mucinous neoplasm. In conclusion, biliary cystic tumors with bile duct communication could be regarded as intraductal papillary neoplasm with a prominent cystic dilatation of the bile duct and mucin retention, rather than true biliary cystic neoplasms.