Kazuyoshi Yamauchi

Natural history of gastric mucosal cytokine expression in Helicobacter pylori gastritis in Mongolian gerbils.

ABSTRACT Data regarding the chronological changes in gastric mucosal cytokines in the different phases of Helicobacter pylori infection are unavailable. We examined Mongolian gerbils for up to 52 weeks after H. pylori (ATCC 43504) inoculation. Levels of mRNAs of mucosal cytokines (interleukin-1β [IL-1β], gamma interferon [IFN-γ], IL-4, IL-6, and IL-10) were assessed using real-time reverse transcription-PCR. Starting 26 weeks after H. pylori inoculation, two clinicohistologic patterns appeared: gastric ulcers in 32% and hyperplastic polyps in 68% of gerbils. High levels of mucosal IL-1β mRNA were observed early in the infection, reaching maximum at 4 weeks and then rapidly declining. Mucosal IFN-γ mRNA also reached maximal levels at 4 weeks but remained high thereafter. Both IL-1β and IFN-γ mRNA levels were consistently higher in the pyloric mucosa than in the fundic mucosa. In contrast, IL-4, IL-6, and IL-10 mRNA levels peaked at 8 to 26 weeks and levels were similar in the pyloric mucosa and the fundic mucosa. IFN-γ mRNA levels were significantly higher in gerbils with ulcers than in those with hyperplastic polyps (median IFN-γ/glyceraldehyde-3-phosphate dehydrogenase ratio × 100,000 = 650 versus 338, respectively [antrum], and 172 versus 40, respectively [corpus]) (P < 0.05). We propose that the different outcomes (e.g., ulcers or hyperplastic polyps) might relate to imbalances among cytokines.

Clinical utility of quantitative RT-PCR targeted to α1,4-N-acetylglucosaminyltransferase mRNA for detection of pancreatic cancer

α1,4‐N‐Acetylglucosaminyltransferase (α4GnT) is a glycosyltransferase responsible for the biosynthesis of α1,4‐GlcNAc‐capped O‐glycans, and is frequently expressed in pancreatic cancer cells but not peripheral blood cells. In the present study, we tested the clinical utility of α4GnT mRNA expressed in the mononuclear cell fraction of peripheral blood as a biomarker of pancreatic cancer. Total RNA isolated from the peripheral blood mononuclear cells from 55 pancreatic cancer patients, 10 chronic pancreatitis patients, and 70 cancer‐free volunteers was analyzed quantitatively by reverse transcription‐polymerase chain reaction with primers specific for α4GnT, and the expression level of α4GnT mRNA relative to that of glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH) was measured. When the ratio of α4GnT to GAPDH transcripts exceeded a defined cut‐off value, patients were considered to have pancreatic cancer. By these standards, 76.4% of the pancreatic cancer patients were detected by this assay. A strong correlation was obtained between positivity in this assay and the expression of α4GnT protein detected immunohistochemically in pancreatic cancer tissues resected subsequently, suggesting that α4GnT mRNA detected in the peripheral blood is derived from circulating pancreatic cancer cells. Although increased levels of α4GnT mRNA was detected in 40.0% of chronic pancreatitis patients and 17.1% of cancer‐free volunteers, the expression levels were significantly lower than those seen in pancreatic cancer patients. These results suggest that quantitative analysis of α4GnT mRNA expressed in the mononuclear cell fraction of peripheral blood will contribute to the detection of pancreatic cancer. (Cancer Sci 2006; 97: 119  – 126)

Quantitative analysis of the effect of Helicobacter pylori on the expressions of SOX2, CDX2, MUC2, MUC5AC, MUC6, TFF1, TFF2, and TFF3 mRNAs in human gastric carcinoma cells

Objective. To investigate the phenotypic characters of carcinoma cells and the response of gastric epithelial cells to Helicobacter pylori (H. pyroli) infection using the gastric carcinoma cell lines. Material and methods. Real-time reverse transcription-polymerase chain reaction (RT-PCR) was used to assess the effect of H. pylori infection on mRNA levels of transcription factors (SOX2 and CDX2), mucin core proteins (MUC2, MUC5AC, and MUC6), and trefoil factor family peptides (TFF) (TFF1, TFF2, and TFF3) in gastric carcinoma cells (AGS, MKN45, and KATO III cells). H. pylori ATCC 43504 and its isogenic cag pathogenicity island (PAI) deleted mutant were used. Results. These cell lines expressed mixed gastric and intestinal phenotypes. The intestinal phenotype predominated in AGS cells and gastric phenotypes in MKN45 and KATO III cells. In all three cell lines, H. pylori infection inhibited SOX2 mRNA expression, but induced the three TFFs mRNAs. In AGS cells, H. pylori induced cag PAI-dependent mRNA expression of CDX2, MUC2, MUC5AC, and MUC6. mRNA expressions of CDX2, MUC5AC, and MUC6 were inhibited in KATO III cells, whereas MUC2 mRNA expression was unchanged. In MKN45 cells, H. pylori induced the three MUCs mRNAs but inhibited CDX2 mRNA expression. Conclusions. This study provides a useful platform for selecting appropriate cell lines to model H. pylori-related changes in the gastric epithelium that mirror the changes seen in vivo. The outcome of H. pylori infection may reflect changes in the mucus gel layer caused by altered expression of mucins and TFF peptides.

t(8;21;14)(q22;q22;q24) is a novel variant of t(8;21) with chimeric transcripts of AML1-ETO in acute myelogenous leukemia

We report a male patient with acute myelogenous leukemia (AML; French-American-British M2) associated with AML1-ETO. Cytogenetic studies showed a complex karyotype including a novel translocation (8;21;14)(q22;q22;q24) in all analyzed cells. This three-way translocation was confirmed with spectral karyotyping. Reverse transcription-polymerase chain reaction analysis for AML1-ETO chimeric transcripts showed the presence of the fusion product with the expected size. Translocation (8;21;14)(q22;q22;q24) is a novel variant of t(8;21)(q22;q22), possibly having a common molecular pathogenetic mechanism.

The Asian project for collaborative derivation of reference intervals: (2) results of non-standardized analytes and transference of reference intervals to the participating laboratories on the basis of cross-comparison of test results

Abstract Background: The 2009 Asian multicenter study for derivation of reference intervals (RIs) featured: 1) centralized measurements to exclude reagent-dependent variations; 2) inclusion of non-standardized analytes (hormones, tumor makers, etc.) in the target; and 3) cross-check of test results between the central and local laboratories. Transferability of centrally derived RIs for non-standardized analytes based on the cross-check was examined. Methods: Forty non-standardized analytes were centrally measured in sera from 3541 reference individuals recruited by 63 laboratories. Forty-four laboratories collaborated in the cross-check study by locally measuring aliquots of sera from 9 to 73 volunteers (average 22.2). Linear relationships were obtained by the major-axis regression. Error in converting RIs using the regression line was expressed by the coefficient of variation of slope b [CV(b)]. CV(b) <10% was set as the cut-off value allowing the conversion. The significance of factors for partitioning RIs was determined similarly as in the first report. Results: Significant sex-, age-, and region-related changes in test results were observed in 17, 15, and 11 of the 40 analytes, respectively. In the cross-comparison study, test results were not harmonized in the majority of immunologically measured analytes, but their average CV(b)s were <10% except for total protein, cystatin C, CA19-9, free thyroxine, and triiodothyronine. After conversion, 74% of centrally derived RIs were transferred to each local laboratory. Conclusions: Our results point to the feasibility of: 1) harmonizing test results across different laboratories; and 2) sharing centrally derived RIs of non-standardized analytes by means of comparative measurement of a set of commutable specimens.

B : b interactions are essential for polymerization of variant fibrinogens with impaired holes 'a'

Background: Fibrin polymerization is mediated by interactions between knobs ‘A’ and ‘B’ exposed by thrombin cleavage, and holes ‘a’ and ‘b’ always present in fibrinogen. The role of A:a interactions is well established, but the roles of knob:hole interactions A:b, B:b or B:a remain ambiguous.Objectives: To determine whether A:b or B:b interactions have a role in thrombin‐catalyzed polymerization, we examined a series of fibrinogen variants with substitutions altering holes ‘a’: γ364Ala, γ364His or γ364Val.Methods: We examined thrombin‐ and reptilase‐catalyzed fibrinopeptide release by high‐performance liquid chromatography, fibrin clot formation by turbidity, fibrin clot structure by scanning electron microscopy (SEM) and factor (F) XIIIa‐catalyzed crosslinking by sodium dodecylsulfate polyacrylamide gel electrophoresis.Results: Thrombin‐catalyzed fibrinopeptide A release was normal, but fibrinopeptide B release was delayed for all variants. The variant fibrinogens all showed markedly impaired thrombin‐catalyzed polymerization; polymerization of γ364Val and γ364His were more delayed than γ364Ala. There was absolutely no polymerization of any variant with reptilase, which exposed only knobs ‘A’. SEM showed that the variant clots formed after 24 h had uniform, ordered fibers that were thicker than normal. Polymerization of the variant fibrinogens was inhibited dose‐dependently by the addition of either Gly‐Pro‐Arg‐Pro (GPRP) or Gly‐His‐Arg‐Pro (GHRP), peptides that specifically block holes ‘a’ and ‘b’, respectively. FXIIIa‐catalyzed crosslinking between γ‐chains was markedly delayed for all the variants.Conclusion: These results demonstrate that B:b interactions are critical for polymerization of variant fibrinogens with impaired holes ‘a’. Based on these data, we propose a model wherein B:b interactions participate in protofibril formation.

Higher avidity binding of apolipoprotein (E–AII) complex than of apolipoprotein E monomer to β‐amyloid

Apolipoprotein E (apoE) is believed to be closely involved in the pathogenesis of Alzheimers disease (AD) because of its ability to bind to β‐amyloid (Aβ), the primary component of senile plaques. The presence of cystein residues in apoE2 and apoE3 allows these isoforms to form disulfide‐linked complexes, such as apo(E–AII) complex and apo(AII–E–AII) complex. A 50‐kDa complex [which corresponded to apo(E–AII)–Aβ, because it reacted with any of the three antibodies, anti‐apoE, anti‐apoAII, or anti‐Aβ] was detected by immunoblot analysis in native cerebrospinal fluid (CSF) obtained from nondementia patients with the apoE phenotype E3/E3. However, a band considered to represent apoE–Aβ was not observed. The dissociation constant (Kd) values obtained for the specific binding of recombinant apoE2, apoE3, and apoE4 to Aβ1–42 were 48.1 ± 2.2 nM, 63.7 ± 2.1 nM, and 75.9 ± 1.8 nM, respectively. In contrast, the binding affinity of the partially purified apo(E3–AII) complex to Aβ1–42 was very high, the Kd being 5.5 ± 0.5 nM. No basic difference was observed between lipidated and nonlipidated apoE in terms of the characteristics of the binding of apoE isoforms to Aβ1–42; however, lipidation reduced the binding capacity of each isoform in a dose‐dependent manner. These findings seem consistent with the generally accepted idea that apoE4 is a risk factor for AD, insofar as only apoE4 is unable to form a complex with apoAII owing to its lack of a cystein residue. In addition, it is possible that apoE3 monomer (and possibly apoE2 monomer), like apoE4 but unlike apo(E–AII) complex, can act as a risk factor in the pathogenesis of AD. J. Neurosci. Res. 58:301–307, 1999.

Specific, rapid, and sensitive enzymatic measurement of sphingomyelin, phosphatidylcholine and lysophosphatidylcholine in serum and lipid extracts.

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.

DNA microarray analysis of T cell-type lymphoproliferative disease of granular lymphocytes

Summary. Lymphoproliferative disease of granular lympho‐ cytes (LDGL) is characterized by the clonal proliferationoflarge granular lymphocytes of either T‐ or natural killer cell origin. To better understand the nature of T cell‐type LDGL, we purified the CD4–CD8+ proliferative fractions from LDGL patients (n=4) and the surface marker‐matched T cells isolated from healthy volunteers (n=4), and compared the expression profiles of 3456 genes using DNA microarray. Through this analysis, we identified a total of six genes whose expression was active in the LDGL T cells, but silent in the normal ones. Interestingly, expression of the gene for interleukin (IL) 1β was specific to LDGL T cells, which was further confirmed by the examination of the serum level of IL‐1β protein. Given its important role in inflammatory reactions, the disease‐specific expression of IL‐1β may have a causative relationship with the LDGL‐ associated rheumatoidarthritis. Spectratyping analysis of the T‐cell receptor repertoire also proved the monoclonal or oligoclonal natureof LDGL cells. These data have shown that microarray analysis with a purified T‐cell subset is an efficient approach to investigate the pathological condition of Tcell‐type LDGL.

Evaluation of the automatic fluorescent image analyzer, Image Titer, for quantitative analysis of antinuclear antibodies.

By making comparisons with the usual manual method, we evaluated an automatic fluorescent image analyzer (Image Titer, Tripath Imaging, Burlington NC), the software for which was developed to simplify measuring indirect immunofluorescent antinuclear antibodies (FANAs). In this new system, images of the stained sample are displayed, and it measures the FANA titer and staining pattern using only 1 slide per subject and does not required the staining of a series of diluted samples as does the manual method. This system showed good reproducibility and linearity for 4 types of control serum samples (with homogeneous, speckled, discrete speckled, and nucleolar staining patterns). In 132 serum samples, consistency between the methods was 100% for the FANA staining pattern and 93.9% for the FANA titer. The Image Titer system detected each pattern in samples with 2 mixed patterns. This system should partly reduce labor and lead to results with minimum differences among individuals, including newly trained persons.