Clinical usefulness of the PAXgene™ bone marrow RNA system for stabilizing total RNA.

The collection of clinical samples, such as bone marrow (BM) and peripheral blood, is an important procedure for the extraction of the cellular RNA. It is essential to preserve the extracted RNA during and after the collection of clinical samples to ensure the accurate analysis of gene expression. To date, the PAXgene™ Blood RNA System has been proven useful for stabilizing RNA extracted from peripheral blood; however, a problem concerning the stability of the total RNA stored using the system has been identified. The PAXgene™ Bone Marrow RNA System (BM system) is a newly developed system, and its clinical usefulness as a stabilizer for the cellular RNA in BM and peripheral blood was investigated with respect to the quality of RNA extracted using this system. A quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was carried out using total RNA extracted with the BM system, which showed that total RNA was more stable in the BM system than in the conventional system, indicating that the BM system can be applied to RT-PCR. The BM system enabled us to detect Wilms' tumor suppressor gene (WT1) more effectively than the conventional system. In conclusion, the BM system is clinically valuable for extracting and stabilizing total RNA of high quality.

Acquired resistance of leukemic cells to AraC is associated with the upregulation of aldehyde dehydrogenase 1 family member A2.

The elucidation of drug resistance mechanisms is important in the development of clinical therapies for the treatment of leukemia. To study the drug resistance mechanisms, protein expression profiles of 1-ß-D-arabinofuranosylcytosine (AraC)-sensitive K562 (K562S) cells and AraC-resistant K562 (K562AC) cells were compared using two-dimensional fluorescence difference gel electrophoresis. In a comparison of protein expression profiles, 2073 protein spots were found to be altered, and 15 proteins of them were remarkably altered. These proteins were identified by mass spectrometry. The most differently expressed proteins were aldehyde dehydrogenase 1 family member A2 (ALDH1A2) and vimentin. Both proteins were verified using reverse transcriptase polymerase chain reaction and Western blot analysis. ALDH1A2 protein was found to be effective in AraC resistance. ALDH1A2 knock-down induced sensitivity to AraC treatment in K562AC cells, and ALDH1A2 overexpressed K562S cells acquired the AraC resistance. Furthermore, the findings also suggest that ALDH1A2 expression is increased after the appearance of AraC resistance in clinical cases. These results will be helpful in understanding the mechanism of AraC resistance.

Inhibition of increased indoleamine 2,3-dioxygenase activity attenuates Toxoplasma gondii replication in the lung during acute infection.

The regulation of local L-tryptophan concentrations by tryptophan-degrading enzyme, indoleamine 2,3-dioxygenase (IDO) induced by various stimuli such as interferon-γ (IFN-γ) is one of the key mechanisms in antimicrobial effect. Recently, IDO is also focused on an immunosuppressive mechanism shared by several different immune cell types. Here, we show that inhibition of increased IDO activity maybe involved in the antiparasitic mechanism during Toxoplasma gondii (T. gondii) infection in vivo. In this study, we investigated the role of IDO by using IDO-gene-deficient (IDO KO) mice and by administering a competitive enzyme inhibitor, 1-methyl-D,L-tryptophan (1MT), to wild-type mice following T. gondii infection. Although depletion of lung l-tryptophan did not occur in IDO KO mice after T. gondii infection, the increased mRNA expression of T. gondii surface antigen gene 2 (SAG2) and the inflammatory cytokines in the lung were drastically reduced in the IDO KO mice following infection. We also found that complete depletion of lung l-tryptophan was observed in wild-type mice after infection, but not in mice treated with 1MT. At the same time, 1MT suppressed the increased mRNA expression of SAG2. Taken together, we observed that the inflammatory damage was significantly decreased by the administration of 1MT in the lung after infection. Inhibition of the IDO activity or the elimination of IDO's substrate may be an effective therapy against microbial diseases.

Effects of PPARγ agonists against vascular and renal dysfunction.

Peroxisome proliferator-activated receptor (PPAR)γ, a nuclear hormone receptor, is activated by its agonists including anti-diabetic thiazolidinediones, and has recently been reported to exert beneficial effects in the vasculature independently of its anti-diabetic effects. We here discuss our recent findings on the beneficial pleiotropic effects of PPARγ agonists. PPARγ agonists have been shown to lower blood pressure in both animals and humans, which may possibly be mediated via the PPARγ agonist-mediated inhibition of the renin-angiotensin-aldosterone system (RAAS) including the suppression of angiotensin (Ang) II type 1 receptor expression/Ang II-mediated signaling pathways and Ang II-induced adrenal aldosterone synthesis/secretion. PPARγ agonists also inhibited the progression of atherosclerosis in both animals and humans. PPARγ agonist-mediated inhibition of the RAAS and the thromboxane A2 system as well as endothelial protection may possibly be involved in the inhibitory effects on blood pressure and atherosclerosis. Furthermore, PPARγ agonists were demonstrated to have reno-protective effects, especially in reducing proteinuria in diabetic nephropathy in both animals and humans. The reno-protective effects of PPARγ agonists were also observed in non-diabetic renal dysfunctions. The effects may possibly be mediated via the PPARγ agonist-mediated blood pressure lowering, endothelial protection, and vasodilation of the glomerular efferent arterioles.. Additionally, anti-neoplastic effects of PPARγ agonists have recently received much attention. PPARγ agonists, may therefore, be useful and effective against lifestyle-related diseases.

[Current update and testing procedures for genetic diagnosis in malignant neoplasm: additional information on pathological tests].

Targeted therapy refers to anticancer treatment which specifically targets key molecules of cancer cells. The higher levels of expression of the epidermal growth factor receptor (EGFR) have also been shown to be correlated with non-small-cell lung cancer (NSCLC) and colorectal cancer (CRC). Potential biomarkers should be investigated for the selection of patients with NSCLC most likely to benefit from epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib. Cetuximab is a monoclonal antibody that binds with high affinity to the EGFR, which is a prime target for new anticancer therapy. However, the predictive value of EGFR expression and mutation of the K-ras gene has been assessed in response to cetuximab. Many molecular techniques are available to assay for these biomarkers. In this review, we present the current assessments of evidence for using these methods as biomarkers for molecular target-based therapy.

WT1 mRNA level in peripheral blood is a sensitive biomarker for monitoring minimal residual disease in acute myeloid leukemia.

The Wilms' tumor gene 1 (WT1) encodes a transcription factor that is involved in normal cellular development and cell survival. WT1 mRNA is overexpressed in the minimal residual disease (MRD) of patients with hematopoietic malignancy patients, particularly acute myeloid leukemia (AML). MRD represents the condition with the low levels of leukemia cells in the bone marrow and is known as a sign of recurrence. In hematopoietic malignancies, definition of remission is based on the lack of MRD at submicroscopic level. Between December 2005 and June 2008, we started to measure WT1 mRNA levels in the peripheral blood (PB) from patients by quantitative real-time PCR in Aomori Prefectural Central Hospital. Three hundreds and eight samples from 95 patients were evaluated. The patients included AML (55 patients), acute lymphoblastic leukemia (11), myelodysplastic syndrome (20), malignant lymphoma (5), chronic myeloid leukemia (1), prostatic carcinoma (1), and leukopenia (2). Among the 55 AML patients, 21 patients were pretreated with remission induction therapy. In the clinical course of 21 patients, timely therapeutic approaches could be started for relapse by the early detection of WT1 mRNA overexpression before the morphological findings were apparent. Monitoring WT1 mRNA is helpful to identify patients at high-risk relapse. High overall survival rate (71.2%, 15/21, median: 24.6 months, range 1.1-35.6 months) was achieved in 3 years. The overall survival rate of 34 post-treatment patients was 61.7% (median: 23.5 months, range 0.13-126.5 months after treatment start). In conclusion, the WT1 mRNA level is a sensitive biomarker for monitoring MRD.

[HCV-RNA test in hepatitis C virus infection--a systematic review].

Hepatitis C virus (HCV) is a major cause of chronic liver disease worldwide. The severity of disease varies widely from mild illness to cirrhosis and hepatocellular carcinoma. The routine diagnostic test for HCV infection is performed using anti-HCV antibodies and an enzyme immunoassay (EIA) for serologic identification and HCV-RNA assay employing the polymerase chain reaction(PCR) for genetic identification. This study assessed the sensitivity and specificity of HCV-RNA diagnostic tests on analysis of the literature. A literature search was performed using the criteria from The standards for Reporting of Diagnostic Accuracy (STARD) steering committee for the assessment of diagnostic tests. The selected studies were searched for identifying publications on the appropriate conducting and reporting of diagnostic studies, and we extracted potential items to generate an extensive list. Out of 409 studies, 38 were selected. Further studies are necessary to accurately access HCV-RNA in various populations compared with reference tests.

[Problems of pre-analysis in genetic testing].

Genetic testing has been developed to confirm various disorders and is applied widely as a fast-growing diagnostic tool. Laboratories performing genetic testing are needed to ensure quality assurance. Accurate and precise testing using nucleic acid extracted from various samples is important as pre-analysis as the results can be affected by bias introduced by sample preparations. The assays estimate the purification and isolation of nucleic acid from samples. Pre-analytic processes such as clinical sampling affected the outcome of genetic testing. Analysis of variance of gene expression revealed small but significant differences between handling methods. Great care has to be taken to measure pre-analytic changes in gene expression. Internal quality control programs for genetic testing are also needed. Thus, well-controlled sample processing and storage conditions are critical for sensitive and potentially quantitative analysis of genetic testing.

[Education and research in the division of biomedical and engineering technology].

The department of Laboratory Science in the Faculty of Medicine at Kyoto University is committed to the development of medicine and medical care for the 21st century. We consider it our mission to contribute to the well being of the entire human race, and we seek students who wish to uphold these ideals. The general field of our Laboratory Medicine may be divided into three major categories; namely, basic and clinical laboratory medicine, biomedical and engineering technology, and medical care for patients and health. Health care relates to bolstering human health from a broad global perspective that encompasses the aspects of environment, health and welfare, and disease prevention.

[Quality assurance of quantitative PCR with real-time reverse transcription polymerase chain reaction method].

The real-time reverse transcription polymerase chain reaction (RT-PCR) method is the most sensitive method for the detection of mRNA and DNA virus. The use of this real-time quantitative PCR(RQ-PCR) has been utilized increasingly to monitor gene expression of many types on clinical diagnosis, and has become standard for the detection and quantification of RNA targets. The employment of the techniques for RQ-PCR offers the advantages of high sensitivity and reproducibility. A number of RQ-PCR have been described that commercial PCR kits are available for quantitative analysis of a limited number of clinically important virus only. Quality assurance is necessary to be assessment the overall process and procedures of quantitative PCR as well as the other clinical testing. Therefore, it should be examined to be used with based on appropriately quality control.

Increased expression of insulin-like growth factor i is associated with Ara-C resistance in leukemia.

Resistance to cytosine arabinoside (Ara-C) is a major problem in the treatment of patients with acute myeloid leukemia (AML). In order to investigate the mechanisms involved in Ara-C resistance, the gene expression profile of Ara-C-resistant K562 human myeloid leukemia cells (K562/AC cells) was compared to that of Ara-C-sensitive K562 cells (K562 cells) by using a cDNA microarray platform. Correspondence analysis demonstrated that insulin-like growth factor I (IGF-I) gene was upregulated in K562/AC cells. The biological significance of IGF-I overexpression was further examined in vitro. When K562 cells were incubated with IGF-I ligand, they were protected from apoptosis induced by Ara-C. In contrast, a significant inhibition of growth and increase of apoptosis of K562/AC cells were induced by IGF-I receptor neutralizing antibody, or suramin, a nonspecific growth factor antagonist. Moreover, from the analysis of 27 AML patients, we have shown that IGF-I expression levels are higher in patients at refractory stage, after Ara-C combined chemotherapy, than those in patients at diagnosis. These results suggest that the inhibition of IGF-I and its downstream pathway is a valuable therapeutic approach to overcome Ara-C resistance in AML.

[New field for the clinical laboratory].

As for clinical inspections, new developments in response to contemporary needs are required during the current rapid medical reforms. Blood inspections now relate to advanced medical fields (such as transplantation, and regeneration medicine), and high skills in these technologies will be in demand from now on. Because of this, it is important to learn the most advanced life engineering technology and to promote blood transfusion inspection, genetic inspection, and so on to enrich it. In particular, bioinfomatics is the field which should be actively advanced because of the tailor-made medical realization, it requires statistical techniques and genome information control studies. Additionally participation in clinical trials (CRC) becomes possible for the execution of the clinical research. These advances are opening up new opportunities in hospitals not only for business expansion as centers for clinical test inspections but also in medicine manufacture, and food company and related inspections. Furthermore, as information network-making that makes the most of medical information and information technology is developed, along with development of the health science field, it is expected that inspection coordinators who build up the relations with hospitals and the area's medical fields will part in the future medical treatment team. Therefore, the maintenance of an educational environment where these skills can be attained is a pressing need so that it may be developed as a new medical field.

Assessment of drug resistance in acute myeloid leukemia.

A major problem in the treatment of leukemia is the development of resistance to chemotherapeutic agents. Assessing the drug resistance of leukemic cells is therefore an important aspect of treatment. One of the main mechanisms of resistance is rapid drug efflux mediated by various members of the ATP-binding cassette transporter superfamily, such as multidrug resistance gene 1 (MDR1), which encodes P-glycoprotein, multidrug resistance-associated protein (MRP) 1 and lung resistance protein. To quantify the degree of acquisition of resistance, several techniques, including drug-sensitivity studies, flow cytometry assay and quantitative gene analysis, have been developed to detect MDR1 and MRP1 gene expression in leukemic cells. However, a significant number of patients may relapse in spite of low expression of MDR1 or MRP1, suggesting the involvement of other intracellular mechanisms, possibly related to cytarabine resistance. This review focuses on the methods aimed at the assessment of drug resistance in acute myeloid leukemia.

Stanniocalcin-1 as a novel marker to detect minimal residual disease of human leukemia.

Stanniocalcin is a glycoprotein hormone that regulates the calcium level in fish. We found that mRNA of human stanniocalcin 1 (STC-1) is detectable in phytohemagglutinin-stimulated T cells and in most human leukemia cell lines, suggesting a role of STC-1 for cell proliferation. This finding prompts us to study the usefulness of STC-1 for monitoring acute leukemia. The levels of STC-1 transcripts increased in patients with acute leukemia at diagnosis and relapse, as judged by quantitative real-time RT-PCR. Levels of transcripts rapidly decreased to within the cut-off levels, when the blast numbers decreased with chemotherapy. Prolonged elevation of STC-1 levels after treatment was associated with a poor prognosis. All of 7 patients relapsed 1 to 4 months after they showed an elevated level of the transcripts in clinical remission. These results indicate that STC-1 is a novel marker for minimal residual disease of acute leukemia, and for an early diagnosis of relapse.

Detection of clone-specific immunoglobulin heavy chain genes in the bone marrow of B-cell-lineage lymphoma after treatment.

In order to determine the appropriate treatment of malignant lymphoma, it is important to know the degree to which extra-nodal invasion of lymphoma cells has occurred. We amplified complementarity-determining region (CDR) III genes in 64% of lymph node samples at the onset or relapse of B-cell-lineage non-Hodgkin's lymphoma (NHL) in 22 patients. By using a clone-specific CDR III probe in each patient, we were able to detect minimal residual disease (MRD) of lymphoma cells in the bone marrow and/or blood in 9 out of 14 cases (64.2%) at the onset of the disease or relapse, whereas abnormal cells in the bone marrow and/or blood were identified by routine morphological analysis in only 4 out of 22 cases (18.2%). This indicates that extranodal invasion of malignant cells may be common in patients with NHL. In some cases, the clone-specific CDR III gene was still expressed in the samples of bone marrow and/or peripheral blood even after chemotherapy, when other markers associated with NHL were no longer expressed. Five out of six cases in this group had a worse outcome associated with NHL. On the other hand, most of the cases whose clone-specific CDR III gene was no longer expressed in the bone marrow and/or in circulation after treatment had a relatively fair prognosis. These results indicate that the detection at molecular level of MRD in extranodal organs may prove useful as a predictor of prognosis for NHL.

Inhibition of motility and invasiveness of renal cell carcinoma induced by short interfering RNA transfection of beta 1,4GalNAc transferase.

Human renal cell carcinoma (RCC) has been characterized by remarkable changes in ganglioside composition. TOS1 cells, typical of metastatic RCC, are characterized by predominance of GM2 as monosialoganglioside, and beta 1,4GalNAc disialyl-Lc(4) (RM2 antigen) as disialoganglioside [J. Biol. Chem. 276 (2001) 16695]. In order to observe the functional role of gangliosides in RCC malignancy, TOS1 cells were transfected with short interfering RNA (siRNA) based on open reading frame sequence of beta 1,4GalNAc transferase (beta 1,4GalNAc-T), and its disordered sequence of siRNA (dsiRNA) as control. In siRNA transfectant, beta 1,4GalNAc-T mRNA level and GM2 expression were greatly reduced, whereby GM3 expression appeared. In contrast, RM2 antigen level was unchanged, even though it has the same beta 1,4GalNAc epitope at the terminus. dsiRNA transfectant showed no change of beta 1,4GalNAc-T mRNA and did not express GM3. Concomitant with reduction of GM2 and appearance of GM3, siRNA transfectant showed greatly reduced motility and invasiveness, although growth rate was unaltered. Both transfectants with siRNA and dsiRNA expressed the same level of tetraspanin CD9. Since CD9/GM3 complex is known to reduce integrin-dependent motility and invasiveness [Biochemistry 40 (2001) 6414], it is plausible that motility and invasiveness of siRNA transfectant of TOS1 cells may be reduced by enhanced formation of such complex.