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Background: In-hospital bleeding is associated with poor prognosis in patients with acute myocardial infarction (AMI). We sought to investigate whether a combination of pre-procedural blood tests could predict the incidence of in-hospital major bleeding in patients with AMI., Methods and Results: A total of 1684 consecutive AMI patients who underwent primary percutaneous coronary intervention (PCI) were recruited and randomly divided into derivation (n = 1010) and validation (n = 674) cohorts. A risk-score model was created based on a combination of parameters assessed on routine blood tests on admission. In the derivation cohort, multivariate analysis revealed that the following 5 variables were significantly associated with in-hospital major bleeding: hemoglobin level < 12 g/dL (odds ratio [OR], 3.32), white blood cell count >10,000/μL (OR, 2.58), platelet count <150,000/μL (OR, 2.51), albumin level < 3.8 mg/dL (OR, 2.51), and estimated glomerular filtration rate < 60 mL/min/1.73 m 2 (OR, 2.31). Zero to five points were given according to the number of these factors each patient had. Incremental risk scores were significantly associated with a higher incidence of in-hospital major bleeding in both cohorts (P < 0.001). Receiver operating characteristic curve analysis of risk models showed adequate discrimination between patients with and without in-hospital major bleeding (derivation cohort: area under the curve [AUC], 0.807; 95% confidence interval [CI], 0.759-0.848; validation cohort: AUC, 0.793; 95% CI, 0.725-0.847)., Conclusions: Our novel laboratory-based bleeding risk model could be useful for simple and objective prediction of in-hospital major bleeding events in patients with AMI., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background/aims: Treating Helicobacter pylori infection in young people is effective for preventing gastric cancer. This study compares the efficacy of triple therapies in adolescents and young adults in Japan., Methods: This multicenter, randomized trial was conducted between February 2012 and March 2015. Infected participants were stratified into adolescents (13-19 years) and young adults (20-39 years). They were randomly assigned to a clarithromycin based (PAC) or metronidazole based (PAM) triple therapy for 1 week., Results: Overall, 137 and 169 participants received the PAC and PAM treatments, respectively. In adolescents, the H. pylori eradication rates were 60.5% and 63.4% for PAC, and 98.3% and 100% for PAM in the intention-to-treat (ITT) and per-protocol (PP) analyses, respectively. In young adults, the eradication rates were 67.0% and 66.7% for PAC, and 95.5% and 96.3% for PAM in ITT and PP analyses, respectively. The eradication rate of PAM was significantly higher than that of PAC in both strata. No severe adverse events were observed., Conclusion: In Japan, PAM may be selected as a first-line treatment for young people with H. pylori if antibiotic susceptibility tests cannot be performed., (Copyright © 2018 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.)

The transcription factor hypoxia-inducible factor-1 (HIF-1) functions as a master regulator of hypoxic response by inducing the transcription of various genes responsible for cellular adaptation to hypoxia. In this study, we investigated the effects of protein inhibitor of activated STAT3 (PIAS3), a small ubiquitin-related modifier (SUMO) E3 ligase, on HIF-1-mediated transcriptional activation. We found that PIAS3 physically associated with HIF-1α. Moreover, PIAS3 overexpression enhanced the transcriptional activity of HIF-1α independently of its SUMO E3 ligase activity. Conversely, quantitative RT-PCR analysis showed that RNAi-mediated PIAS3 knockdown reduced the expression of HIF-1 target genes under hypoxia. In addition, PIAS3 knockdown induced the destabilization of HIF-1α protein, and the destabilization was reversed by the proteasome inhibitor MG132. Taken together, these results suggest that PIAS3 functions as a positive regulator of HIF-1α-mediated transcription by increasing its protein stability., (Copyright © 2015 Elsevier Inc. All rights reserved.)

DNA methyltransferase 1 (Dnmt1) is essential for the maintenance of hematopoietic and somatic stem cells in mice; however, its roles in human cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are still elusive. In the present study, we investigated DNMT1 functions in the maintenance of human colon CSCs/CICs using the human colon cancer cell line HCT116 (HCT116 w/t) and its DNMT1 knockout cell line (DNMT1(-/-)). The rates of CSCs/CICs were evaluated by side population (SP) analysis, ALDEFLUOR assay and expression of CD44 and CD24. SP, ALDEFLUOR-positive (ALDEFLUOR(+)) and CD44-positive and CD24-positive (CD44(+)CD24(+)) cell rates were lower in DNMT1(-/-) cells than in HCT116 w/t cells. Since CSCs/CICs have higher tumor-initiating ability than that of non-CSCs/CICs, the tumor-initiating abilities were addressed by injecting immune deficient (NOD/SCID) mice. DNMT1(-/-) cells showed less tumor-initiating ability than did HCT116 w/t cells, whereas the growing rate of DNMT1(-/-) cells showed no significant difference from that of HCT116 cells both in vitro and in vivo. Similar results were obtained for cells in which DNMT1 had been transiently knocked-down using gene-specific siRNAs. Taken together, these results indicate that DNMT1 is essential for maintenance of colon CSCs/CICs and that short-term suppression of DNMT1 might be sufficient to disrupt CSCs/CICs., (Copyright © 2012 Elsevier Inc. All rights reserved.)

AMP-activated protein kinase (AMPK) is a hetero-trimeric Ser/Thr kinase composed of a catalytic α subunit and regulatory β and γ subunits; it functions as an energy sensor that controls cellular energy homeostasis. In response to an increased cellular AMP/ATP ratio, AMPK is activated by phosphorylation at Thr172 in the α-subunit by upstream AMPK kinases (AMPKKs), including tumor suppressor liver kinase B1 (LKB1). To elucidate more precise molecular mechanisms of AMPK activation, we performed yeast two-hybrid screening and isolated the complementary DNA (cDNA) encoding the nuclear protein Artemis/DNA cross-link repair 1C (DCLRE1C) as an AMPKα2-binding protein. Artemis was found to co-immunoprecipitate with AMPKα2, and the co-localization of Artemis with AMPKα2 in the nucleus was confirmed by immunofluorescence staining in U2OS cells. Moreover, over-expression of Artemis enhanced the phosphorylation of AMPKα2 and the AMPK substrate acetyl-CoA carboxylase (ACC). Conversely, RNAi-mediated knockdown of Artemis reduced AMPK and ACC phosphorylation. In addition, Artemis markedly increased the physical association between AMPKα2 and LKB1. Taken together, these results suggest that Artemis functions as a positive regulator of AMPK signaling by stabilizing the LKB1-AMPK complex., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Objective: Glucagon-like peptide 1 (GLP-1), one of the incretin hormones, has been reported to increase positive inotropic activity in cardiac myocytes and protect against myocardial injury. However, the effects upon endothelial cells and the mechanisms involved are not fully understood. We assessed the hypothesis that GLP-1 has protective effects against inflammation and oxidative stress on human endothelial cells., Methods and Results: The effects of the GLP-1 analog liraglutide upon TNF-α-induced injury of the human umbilical vein endothelial cells (HUVECs) were evaluated. First, ROS induced by TNF-α was measured by staining with CM-H(2)DCFDA. Intracellular ROS production of HUVECs was significantly decreased in a dose-dependent manner until 30 nM while liraglutide inhibited the induction of gp91(phox) and p22(phox), subunit of NADPH oxidase, by TNF-α. In addition, protein levels of SOD-2, catalase and GPx were significantly increased by liraglutide. Second, rapid translocation of PKC-α into the membrane following TNF-α was evident. Liraglutide significantly inhibited this very rapid TNF-α-induced translocation of PKC-α into membrane at 2.5 min. Third, liraglutide significantly inhibited NF-κB activation and upregulated I-κB family while phosphorylation of IKK-α/β, which is upstream of NF-κB signaling, was also downregulated after 15 min of TNF-α treatment. Finally, liraglutide inhibited apoptosis of HUVEC and expression of Pentraxin-3 induced by TNF-α., Conclusion: Liraglutide exerts marked anti-oxidative and anti-inflammatory effects on endothelial cells with inhibition of PKC-α, NADPH oxidase, NF-κB signaling and upregulation of protective anti-oxidative enzymes., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Rikkunshito (RKT), a Japanese traditional medicine, has been shown to stimulate food intake in rats with cisplatin-induced anorexia; however, the underlying mechanisms remain unknown. In this study, we investigated whether RKT is involved in the degradation of peripheral ghrelin. RKT inhibited decreases in plasma ghrelin level and enhanced acyl- to desacyl-ghrelin (A/D) ratio in cisplatin-treated rats. Several components of RKT demonstrated inhibitory activity against ghrelin deacylating enzymes. In addition, 10-gingerol, a component of RKT, inhibited exogenous ghrelin deacylation. Therefore, RKT may enhance plasma acyl-ghrelin level, at least in part, by inhibiting the circulating ghrelin degrading enzyme., (Copyright © 2011 Elsevier Inc. All rights reserved.)

In our previous study, we demonstrated that a peptide derived from the novel centrosome residing protein Cep55/c10orf3 can be targeted by the cytotoxic T lymphocytes (CTLs) in peripheral blood mononuclear cells (PBMCs) of breast carcinoma patients. In this report, we evaluated the feasibility of cancer immunotherapy using Cep55/c10orf3 peptide for colorectal carcinoma (CRC). To evaluate the expression of Cep55/c10orf3 in CRC tissues, we performed immunohistochemical staining of using anti-Cep55/c10orf3 monoclonal antibody. Sixty-three percent cases showed weak positive for Cep55/c10orf3 in total 70 CRC cases. The Cep55/c10orf3 expression intention was collated with high histological grade of CRC. Thus, we hypothesized that Cep55/c10orf3 can also be the target of CTLs in CRC cases. We generated CTLs from PBMCs of human leukocyte antigen (HLA)-A24-positive colorectal carcinoma patients using HLA-A24-restricted Cep55/c10orf3 peptides. Two of 6 colorectal cancer patients were reactive for the Cep55/c10orf3&#95;193(10) peptide, which was the only immunogenic peptide in breast carcinoma patients. CTL clone specific for Cep55/c10orf3&#95;193(10) recognized and lysed HLA-A24 (+) and Cep55/c10orf3 (+) colorectal carcinoma cell lines. In addition, 1 of 6 colorectal carcinoma patients was reactive for the Cep55/c10orf3&#95;402(11) and Cep55/c10orf3&#95;283(12) peptides, but not for Cep55/c10orf3&#95;193(10) with the ELISPOT assay. These observations suggest that the antigenic peptide repertoire presented by HLA-A24 in colorectal carcinoma might be different from that in breast carcinoma. Thus, these peptide vaccination peptide mixture of Cep55/c10orf3&#95;193(10), Cep55/c10orf3&#95;402(11) and Cep55/c10orf3&#95;283(12) might be more effective than a single peptide in the treatment of colorectal carcinoma patients., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The ubiquitin-proteasomal system plays a crucial role in oncogenesis in colorectal tissues. Recent studies have shown that stability of β-catenin, which functions as an oncogene for colorectal cancer, is regulated by ubiquitin-mediated degradation. It has been reported that a putative E3 ubiquitin ligase, RNF43, is highly expressed in human colorectal carcinoma and that RNF43 promotes cell growth. However, the involvement of RNF43 in carcinogenesis has not been fully elucidated. In this study, we found by using yeast two-hybrid screening that RNF43 binds to NEDD-4-like ubiquitin-protein ligase-1 (NEDL1), which enhances pro-apoptotic activity by p53. In addition, we found that RNF43 also interacts with p53 and that RNF43 suppresses transcriptional activity of p53 in H1299 cells and attenuates apoptosis induced by ultraviolet irradiation. These findings suggest that RNF43 is associated with p53-mediated apoptosis in collaboration with NEDL1 in colorectal carcinogenesis., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: The relation between Helicobacter pylori infection and gastric cancer has been proven in epidemiological studies and animal experiments. Our aim was to investigate the prophylactic effect of H pylori eradication on the development of metachronous gastric carcinoma after endoscopic resection for early gastric cancer., Methods: In this multi-centre, open-label, randomised controlled trial, 544 patients with early gastric cancer, either newly diagnosed and planning to have endoscopic treatment or in post-resection follow-up after endoscopic treatment, were randomly assigned to receive an H pylori eradication regimen (n=272) or control (n=272). Randomisation was done by a computer-generated randomisation list and was stratified by whether the patient was newly diagnosed or post-resection. Patients in the eradication group received lansoprazole 30 mg twice daily, amoxicillin 750 mg twice daily, and clarithromycin 200 mg twice daily for a week; those in the control group received standard care, but no treatment for H pylori. Patients were examined endoscopically at 6, 12, 24, and 36 months after allocation. The primary endpoint was diagnosis of new carcinoma at another site in the stomach. Analyses were by intention to treat. This trial is registered with the UMIN Clinical Trials Registry, number UMIN000001169., Findings: At 3-year follow-up, metachronous gastric carcinoma had developed in nine patients in the eradication group and 24 in the control group. In the full intention-to-treat population, including all patients irrespective of length of follow-up (272 patients in each group), the odds ratio for metachronous gastric carcinoma was 0.353 (95% CI 0.161-0.775; p=0.009); in the modified intention-to-treat population, including patients with at least one post-randomisation assessment of tumour status and adjusting for loss to follow-up (255 patients in the eradication group, 250 in the control group), the hazard ratio for metachronous gastric carcinoma was 0.339 (95% CI 0.157-0.729; p=0.003). In the eradication group, 19 (7%) patients had diarrhoea and 32 (12%) had soft stools., Interpretation: Prophylactic eradication of H pylori after endoscopic resection of early gastric cancer should be used to prevent the development of metachronous gastric carcinoma., Funding: Hiroshima Cancer Seminar Foundation.

Background: The acceptance of the premise that Helicobacter pylori infection is aetiologically related to gastric cancer and peptic ulcer and that the risk of gastric cancer among Helicobacter pylori infected individuals is related to the extent, severity and duration of atrophic gastritis has led to major changes in medical and endoscopic practices. The development of non-invasive methods to detect Helicobacter pylori and to estimate the extent and severity of gastritis has reduced the need for diagnostic endoscopy in asymptomatic individuals., Methods and Results: Here we provide recommendations regarding deciding whether non-invasive and endoscopic assessment of the gastric mucosa is preferred. We also include specific recommendations and caveats regarding the preferred biopsy number and sites as well as the identification of specimens, to allow the pathologist to reliable stage the severity and extent of gastritis, and thus provide prognostic information needed for patient managements (e.g., whether endoscopic surveillance is recommended)., Conclusion: In summary, while there is clearly a role for gastric endoscopy and endoscopic biopsy in the Helicobacter pylori era, obtaining useful diagnostic and prognostic information is critically dependent upon attention to detail with regard to biopsy site and identification as to the location from where the specimen was taken.

Background: There have been several reports describing a notable hyperamylasaemia in patients with multiple myeloma. Such amylase-producing myelomas have been mainly described in the context of concomitant salivary-type hyperamylasaemia, with sialyl salivary-type amylase identified in a portion of those cases. We investigated the incidence of the production of sialyl salivary-type amylase in serum of multiple myeloma patients., Methods: Eleven patients (6 male and 5 female) who had been diagnosed as having multiple myeloma were enrolled in this study. Sialyl salivary-type amylase was detected by isoamylase electrophoresis and HPLC analysis, and identified by detecting either abnormal neuraminidase-sensitive band through isoamylase electrophoresis or abnormal extra-elution peak of amylase by means of HPLC analysis., Results: Sialyl salivary-type amylase was detected in 7 out of 11 (63.6%) patients. Median total amylase activity was 154 U/l (range 109-43020). Isoamylase electrophoretic patterns of patients' serum were normal in 5 patients (71.4%) out of 7 patients and salivary-dominant in 2 (50.0%) out of 4 patients., Conclusions: We consider that there is no significant relationship between total serum amylase level and amylase isoenzyme pattern in the incidence of production of sialyl salivary-type amylase with multiple myeloma.

MALT1, BCL10 (B-cell lymphoma 10), and API2 (apoptosis inhibitor 2)-MALT1 are key molecules in mucosa-associated lymphoid tissue (MALT) lymphomagenesis. We previously reported that MALT1 and API2-MALT1 were localized only in cytoplasm, where we suggested that both molecules were likely to be active. In the study presented here, we further examined the localization-determining region by generating various mutants and were able to demonstrate that there were nuclear export signal (NES)-containing domains in the MALT1 C-terminal region. The use of leptomycin B, an NES-specific inhibitor, demonstrated that both MALT1 and API2-MALT1 were predominantly retained in the nuclei, indicating that these molecules were shuttling between nucleus and cytoplasm in an NES-dependent manner. It was also found that MALT1 was involved in the nuclear export of BCL10, which is originally localized in both nucleus and cytoplasm. These results correlate well with the nuclear BCL10 expression pattern in both t(1;14) and t(11;18) MALT lymphomas. The nucleocytoplasmic shuttling of MALT1 and BCL10 complex may indicate that these molecules are involved not only in the nuclear factor kappaB (NF-kappaB) pathway but also in other biologic functions in lymphocytes.

Proteolytic modification of insulin-like growth factor binding proteins (IGFBPs) plays an important physiological role in regulating insulin-like growth factor (IGF) bioavailability. Recently, we demonstrated that matrix metalloproteinase-7 (MMP-7)/Matrilysin produced by various cancer cells catalyzes the proteolysis of IGFBP-3 in vitro and regulates IGF bioavailability, resulting in an anti-apoptotic effect against anchorage-independent culture. In the present study, we investigated whether MMP-7 contributes to proteolysis of the other five IGFBPs, IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5, and IGFBP-6, and whether this results in phosphorylation of the IGF type 1 receptor (IGF-1R). MMP-7 cleaved all six IGFBPs, resulting in IGF-mediated IGF-1R phosphorylation, which was inhibited by EDTA treatment. These results suggest that MMP-7 derived from cancer cells can regulate IGF bioavailability in the microenvironment surrounding the tumor, where various kinds of IGF/IGFBP complexes are found, thereby favoring cancer cell growth and survival during the processes of invasion and metastasis.

In clinical Helicobacter pylori isolates, metronidazole resistance has been associated with mutations in the rdxA and frxA genes. The aim of this study was to examine the role of the rdxA and frxA genes after the in vitro induction of metronidazole resistance. A total of five suscep-tible H. pylori isolates were initially exposed to different subinhibitory metronidazole concentrations to induce in vitro resistance to metronidazole. Susceptible and resistant strains after the in vitro induction of resistance were examined to evaluate mutations of the rdxA and frxA genes by sequence analysis. After the in vitro induction of resistance, analysis revealed that two and four susceptible strains developed resistance when cultured with 0.3 microg/ml and 0.6 microg/ml of metronidazole, respectively. Before and after the induction of resistance, none of the susceptible strains that developed low and moderate levels of resistance presented any mutation in either of the evaluated genes, whereas strains with high-level metronidazole resistance contained a simple mutation of the frxA gene, but no specific changes in the rdxA gene. Strains with moderate-level resistance contained both single and multiple mutations of rdxA and frxA, respectively, and the low-level-metronidazole-resistant strain contained a single mutation in the frxA gene, without any significant change in the rdxA gene. In this study, the strains that developed resistance were mainly associated with mutations of the frxA gene, suggesting the possibility that inactivation of this gene could originate metronidazole resistance. The results after the in vitro induction of resistance to metronidazole suggested the presence of additional metronidazole resistance mechanisms, other than mutations of the rdxA and/or frxA genes.

Background/aims: The molecular basis of the metastasis of hepatocellular carcinoma (HCC) is not fully understood. The aim of this study was to elucidate the crucial genes involved in metastasis of HCC., Methods: We compared expression profiles among highly metastatic HCC cell lines and non-metastatic HCC cell lines by using oligonucleotide array to identify genes associated with metastasis. We further investigated the effect of identified gene on cell motility and metastasis in vitro and in vivo. Finally, we examined immunohistochemistry in human tissue samples., Results: We identified 39 genes whose expression levels were significantly correlated with metastatic ability (P<0.05). Of these genes, we further investigated cortactin, because this cortical actin-associated protein is a substrate of Src, whose activation has been shown to be involved in HCC cell migration and metastasis. Overexpression of cortactin in a non-metastatic HCC cell line increased cell motility, and resulted in metastasis in an orthotopic model. Furthermore, immunohistochemical expression of cortactin revealed its significant overexpression in HCC with intrahepatic metastasis compared with HCC without intrahepatic metastasis (P<0.005)., Conclusions: Overexpression of cortactin may play a role in the metastasis of HCC by influencing cell motility, and cortactin could be a sensitive marker for HCC with intrahepatic metastasis.

Inhibitory natural killer cell receptor (NKR)-expressing cells may induce a graft-versus-leukemia/tumor (GVL/T) effect against leukemic cells and tumor cells that have mismatched or decreased expression of HLA class I molecules and may not cause graft-versus-host disease (GVHD) against host cells that have normal expression of HLA class I molecules. In our study, we were able to expand inhibitory NKR (CD94/NKG2A)-expressing CD8+ T cells from granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (G-PBMCs) by more than 500-fold using stimulation by an anti-CD3 monoclonal antibody with interleukin 15 (IL-15). These expanded and purified CD94-expressing cells attacked various malignant cell lines, including solid cancer cell lines, as well as the patients' leukemic cells but not autologous and allogeneic phytohemagglutinin (PHA) blasts in vitro. Also, these CD94-expressing cells prevented the growth of K562 leukemic cells and CW2 colon cancer cells in NOD/SCID mice in vivo. On the other hand, the CD94-expressing cells have low responsiveness to alloantigen in mixed lymphocyte culture (MLC) and have high transforming growth factor (TGF)-beta1- but low IL-2- producing capacity. Therefore, CD94-expressing cells with cytolytic activity against the recipient's leukemic and tumor cells without enhancement of alloresponse might be able to be expanded from donor G-PBMCs.

Krüppel-like factors (KLFs) are key transcriptional regulators of cell differentiation and proliferation. Among the KLF family, the expression of KLF4 (GKLF) and KLF5 (IKLF) is highly restricted in the epithelial cells of several organs such as the gut and skin, and it has been reported that these epithelial-type KLF genes may be involved in colon carcinogenesis. Recently we found that Klf4 and Klf5 genes were significantly expressed in the developmental bladder epithelium of mice as well. Therefore, in this report we studied the involvement of the KLF4 and KLF5 genes in bladder carcinogenesis. First, we analyzed the expression of KLF4 and KLF5 in a variety of human bladder cancer cell lines and surgical specimens by RNA blot and in situ hybridization analyses. Both genes were highly expressed in the normal bladder epithelium, whereas KLF4, but not KLF5, was frequently downregulated in bladder cancer cell lines and cancer tissues. We then transduced the KLF4 and KLF5 genes into the bladder cancer cell lines using adenoviral vectors to examine the biological activities of the genes on those cells. The transduction of KLF4, but not KLF5, suppressed cell growth and induced apoptosis. Our study suggests that inactivation of KLF4 is one of the frequent steps towards bladder carcinogenesis.

In the tumor cells exposed to hypoxia, hypoxia-inducible factor-1 (HIF-1)-mediated adaptation responses such as angiogenesis and anaerobic metabolism are induced for their survival. We have recently reported that the constitutive expression of HIF-1 alpha renders pancreatic cancer cells resistant to apoptosis induced by hypoxia and glucose deprivation. We then established dominant-negative HIF-1 alpha (dnHIF-1 alpha) transfectants and examined their susceptibility to apoptosis and growth inhibition induced by hypoxia and glucose deprivation in vitro and their tumorigenicity in SCID mice. We further examined the expressions of aldolase A and Glut-1 in vitro and Glut-1 expression and glucose uptake in the tumor tissues and microvessel counts in the tumor tissues. As a result, dnHIF-1 alpha rendered the pancreatic cancer cells sensitive to apoptosis and growth inhibition induced by hypoxia and glucose deprivation. Also it abrogated the enhanced expression of Glut-1 and aldolase A mRNAs under hypoxia and reduced the expression of Glut-1 and the glucose uptake in the tumor tissues and consequently in vivo tumorigenicity. We found no significant difference in the microvessel counts among the tumor tissues. From these results, we suggest that the disruption of the HIF-1 pathway might be effective in the treatment of pancreatic cancers.

Several studies have revealed that the Helicobacter pylori genome differs markedly from strain to strain, perhaps as a result of mutations arising during persistent infection and/or related to the observed variation in virulence. The development of a detection system for mutations in H. pylori genes might therefore help us to develop a better understanding of its mutability, and in this way help us to develop plans for investigating the relationship between its genomic variability and the pathogenesis of various gastric and duodenal diseases associated with the long-term H. pylori infections. We have therefore begun a study of H. pylori mutability using the endogenous rpsL gene as a marker. Spontaneous mutant frequencies were measured and compared among H. pylori strains, after incubation on plates containing 50 microg/ml of streptomycin for 10 days as a selection procedure. The rpsL gene of each streptomycin-resistant (Str(r)) mutant was amplified by polymerase-chain-reaction (PCR) and sequenced. All of the mutations we characterized were localized at codons 43 or 88 of the rpsL gene and were base transitions from A to G, replacing lysine with arginine. This is in contrast to the spontaneous Str(r) mutants isolated from Escherichia coli, which resulted from either A to G transitions at lysine codons 42 and 87, or A to T or C transversions at lysine codon 42. The spontaneous mutant frequencies of the rpsL gene in H. pylori were of the order of 10(-9), and there were significant differences in spontaneous mutant frequencies among the strains tested. This mutation detection system might be of value in screening clinical isolates for H. pylori mutator phenotypes.

Whereas mobilization to inflammatory sites is an important function of neutrophils, it remains to be determined whether granulocyte colony-stimulating factor (G-CSF) stimulates the mobilization of neutrophils to the inflammatory sites. This study compared the expression of more than 9000 genes in neutrophils treated with and without G-CSF with the use of a DNA microarray system to determine the effects of G-CSF on the function of neutrophils. It was found that messenger RNA expression of epithelial cell-derived neutrophil attractant-78 (ENA-78), which has been reported to be a chemotactic factor for neutrophils, was induced by G-CSF in neutrophils. The study demonstrated that the supernatant of G-CSF-treated neutrophils induced the chemotaxis of neutrophils and that anti-ENA-78 antibody and anti-CXCR-2 antibody inhibited the chemotaxis. These data suggest that G-CSF may enhance the mobilization of neutrophils and consequently augment the accumulation of neutrophils in the inflammatory sites through the secretion of ENA-78.

To elucidate the mechanisms by which hematopoietic progenitor cells transmigrate via the bone marrow (BM) endothelial cells, we first established endothelial cell lines from BM and lung, and BM fibroblast cell lines; then we established an in vitro model of transendothelial migration of hematopoietic progenitor cells in the presence of chemoattractants secreted by BM fibroblast cells. The BM endothelial cells expressed vascular cell adhesion molecule-1 (VCAM-1), but the lung endothelial cells did not. The BM fibroblast cells secreted chemoattractants including stroma cell-derived factor (SDF)-1, which could attract hematopoietic progenitor cells to BM and activate the adhesion molecules expressed on hematopoietic progenitor cells after rolling along the endothelial cells. Anti-SDF-1 antibody inhibited the transendothelial migration of a hematopoietic progenitor cell line, FDCP-2. FDCP-2 that expressed very late activation antigen-4 (VLA-4) and normal progenitor cells transmigrated through BM endothelial cells but not lung endothelial cells, even if in the presence of chemoattractants produced by BM fibroblasts. Both anti-VLA-4 and anti-VCAM-1 antibodies inhibited the transendothelial migration of FDCP-2 cells and normal hematopoietic progenitor cells. These findings suggest that the transendothelial migration of hematopoietic progenitor cells is characteristic of BM endothelial cells, and that VLA-4/VCAM-1 and SDF-1 play important roles in the transendothelial migration and, consequently, homing of hematopoietic progenitor cells to BM.

Familial essential thrombocythemia (ET) is inherited in an autosomal-dominant manner. This finding implies that familial ET may arise as a consequence of a mutation(s) that activates platelet production. In 1994, the thrombopoietin (TPO) gene was isolated and cloned. The TPO-TPO receptor, encoded for by the c-mpl gene, are essential regulators of thrombopoiesis. Alterations of TPO or c-Mpl thus may constitute a pathogenic event leading to familial ET. In a case of familial ET presented in our institute, serum TPO levels were significantly elevated in affected members of the family as compared with nonaffected members. Moreover, we identified a one-base deletion in the 5'-untranslated region of the TPO gene in affected but not in nonaffected family members. In vitro experiments showed that the identified mutation increased TPO production. Based on our findings, we propose that this region of the TPO gene may play a crucial role in regulating TPO expression. Our results strongly suggest that the identified mutation leads to familial ET., (Copyright 1998 by The American Society of Hematology.)

A subunit specific radioimmunoassay was developed for the quantification of human aldolase A, B, and C. The method used was a double antibody radioimmunoassay using radioiodinated purified aldolase A, B, or C subunits as the ligand, specific chicken antibodies to aldolase isozymes and rabbit antibodies to chicken IgG. The Iodogen method was used for iodination of the purified isozyme subunits in this study. Human brain tissue contained similar concentrations of aldolase A and aldolase C, and a smaller amount of aldolase B, which was the main isozyme of liver tissue. Levels of serum aldolase A were greater than 203 ng/ml, the upper limit of normal, in six of 24 patients with cerebral infarction and in 11 of 31 patients with cerebral hemorrhage. Nine of 24 patients with cerebral infarction and 16 of 31 patients with cerebral hemorrhage had serum aldolase C levels greater than 4.1 ng/ml, the upper limit in normal sera. These data suggest that serum aldolase C may be a more specific and sensitive marker of cerebrovascular diseases than aldolase A. We also demonstrated that serial measurement of serum aldolase C in patients with cerebrovascular diseases might be useful in estimating prognosis, since serially increasing serum aldolase C levels during the course of these diseases were correlated with a high mortality rate.

A solid-phase, non-competitive radioimmunoassay for aldolase A in human serum has been developed. Human aldolase A was purified from muscle, and specific antisera to the purified aldolase A were obtained from chickens. Specific IgG anti-human aldolase A was purified by affinity chromatography. Disposable polypropylene plates were coated with specific IgG antibody and used for radioimmunoassay with 125I-specific IgG antibody to aldolase A. The non-specific binding was minimized by saturating the binding sites of the plates with 2% ovalbumin in 0.1% Tween 20. This radioimmunoassay is specific for the aldolase A subunit, with no cross-reactivity with human aldolase B subunit or homopolymeric human aldolase C(C4). The serum aldolase A immunoreactivities of 33 normal subjects ranged from 124 to 212 ng/ml with a mean of 178 +/- 41 ng/ml (+/- 2 SD). Ninety-three patients' sera were assayed with both a solid-phase non-competitive radioimmunoassay and a competitive double antibody radioimmunoassay developed in our laboratory and the results showed a high degree of correlation (r = 0.912; p less than 0.001). Rapidity and simplicity of the solid-phase assay makes it superior to other methods for the measurement of serum aldolase isozymes.

A radioimmunoassay was developed for the direct quantification of aldolase A in human serum. The method is a double antibody radioimmunoassay using radioiodinated aldolase A4 homopolymer as ligand, chicken antibodies to aldolase A, and rabbit antibodies to chicken IgG. The lowest measurable amount by this method was 2 ng (0.01 U). The radioimmunoassay was shown to be specific for the aldolase A subunit, with no cross-reactivity with human aldolase B subunits or homopolymeric human aldolase C (C4). The immunoreactive aldolase A in the sera of 41 normal healthy subjects ranged from 130 to 210 ng/ml (0.81-1.31 U/1), with a mean of 171 /+- 39 ng/ml.

A radioimmunoassay was developed for the direct quantification of aldolase B in human serum and tissues. The method is a double antibody radioimmunoassay technique using radioiodinated aldolase B homopolymer as ligand, chicken antibodies to aldolase B and rabbit antibodies to chicken IgG. This radioimmunoassay was shown to be specific for the aldolase B subunit, with no cross-reactivity with either human aldolase A subunit or homopolymeric human aldolase C (C4). The lowest measurable amount by this method was 2 ng/ml. Aldolase B is predominantly found in normal liver tissue, with relatively high aldolase B levels also observed in kidney. Aldolase B levels in the serum obtained from 11 normal subjects ranged from 23 to 38 ng/ml, with a mean of 28.5 +/- 9.2 (S.D.) ng/ml. Almost all of patients with hepatitis had serum aldolase B levels greater than 30 ng/ml. In cancer patients, serum aldolase B was slightly elevated in patients with metastatic liver cancer and primary lever cell carcinoma, whereas no elevation of serum aldolase B was shown in patients without liver metastasis.