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.

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.

[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.

[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.

[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.

[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.

Alteration in copy numbers of genes as a mechanism for acquired drug resistance.

Chemoresistance is a major obstacle for successful treatment of cancer. To identify regions of the genome associated with acquired resistance to therapeutic drugs, we conducted molecular cytogenetic analyses of 23 cancer-cell lines, each resistant to either camptothecin, cisplatin, etoposide (VP-16), Adriamycin, or 1-beta-D-arabinofuranosylcytosine, although the parental tumor lines were not. Subtractive comparative genomic hybridization studies revealed regions of gain or loss in DNA-copy numbers that were characteristic of drug-resistant cell lines; i.e., differences from their drug-sensitive parental cell lines. Thirteen ATP-binding cassette (ABC) transporter genes [ABCA3, ABCB1 (MDR1), ABCB6, ABCB8, ABCB10, ABCB11, ABCC1 (MRP1), ABCC4, ABCC9, ABCD3, ABCD4, ABCE1, and ABCF2] were amplified among 19 of the resistant cell lines examined. Three genes encoding antiapoptotic BCL-2 proteins (BCL2L2, MCL1, and BCL2L10) were also amplified and consequently overexpressed in three of the derivative lines. Down-regulation of BCL2L2 with an antisense oligonucleotide sensitized a VP-16 resistant ovarian-cancer cell line (SKOV3/VP) to VP-16. A decrease in copy numbers of genes encoding deoxycytidine kinase, DNA topoisomerase I, and DNA topoisomerase II alpha reduced their expression levels in one cytosine arabinoside-resistant line, two of three camptothecin-resistant lines, and two of five VP-16-resistant cell lines, respectively. Our results indicated that changes in DNA-copy numbers of the genes mentioned can activate or down-regulate them in drug-resistant cell lines, and that such genomic alterations might be implicated in acquired chemoresistance.

[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.

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.

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.

Abnormal white matter lesions with sensorineural hearing loss caused by congenital cytomegalovirus infection: retrospective diagnosis by PCR using Guthrie cards.

We report on two patients with congenital cytomegalovirus (CMV) infection asymptomatic at birth that was diagnosed retrospectively by polymerase chain reaction (PCR) of CMV DNA using blood stored on Guthrie cards. Neuroimaging studies showed abnormal myelination without any gray matter abnormalities. In the differential diagnosis of patients with abnormal white matter lesions and sensorineural hearing loss, one should consider congenital CMV infection. When investigating the etiology of patients with behavioral problems, migrational disorder, or white matter disease, PCR analysis of CMV DNA using blood stored on Guthrie cards might be helpful.

[A novel cDNA array method for detecting the chimeric genes in human leukemia].

Advanced molecular diagnostics in hematological malignancy have provided us with a broad assortment of new assays and techniques. We have developed a simple cDNA microarray method for detecting the chimeric genes in human leukemia. The method, based on the biotin-avidine reaction, is performed with color development. The oligotip we have developed is indicated by simple and highly sensitive data. By analyzing with this method, we can obtain useful information about the subtype of chimeric genes in leukemia.

[Molecular diagnosis for diagnosis of leukemia].

Molecular methods are emerging as important tools for diagnosis and therapy of patients with leukemia. First, the development of conventional Southern blot analysis has facilitated the detection of rearrangements in immunoglobulin and T-cell receptor genes. Moreover, many chimeric genes involved in balanced translocation have identified significant classifications of leukemia patients by cytogenetic techniques such fluorescent in situ hybridization (FISH) and reverse-transcriptase polymerase chain reaction (RT-PCR). These techniques provide an advantage in monitoring minimal residual disease (MRD). Furthermore, recent gene expression analysis with quantitative PCR assays such as real-time PCR have developed the early diagnosis and monitoring of MRD. These molecular-based diagnoses have contributed to the clinical decision-making process in the diagnosis of leukemia.

[Genetic education in pre- and post-graduation].

Molecular genetics is now integral to all aspects of biomedical science. Every medical school should have a best program of basic and practical genetics education as one of the core curriculum. Genetic information includes DNA, chromosome analysis and clinical tests, as well as family history. The benefit of patient received genetic tests is counseling by counselors well educated. Thus each medical and technological school must find the best way to incorporate genetics teaching into its own curriculum.

[The ethical implications, guidelines, and standardization of genetic tests].

Laboratory advances in molecular genetics have resulted in numerous clinical applications for DNA analysis. Genetic tests can contribute a great deal of information to clinical diagnosis. These genetic tests including PCR, fluorescence detection, real-time PCR, and automated sequencing have developed into both simple and time-consuming laboratory techniques. Currently, DNA diagnosis is not used routinely because of cost, complexity, and resources. We have focused on the ethical implications and proper standardization in DNA diagnosis. This review is to discuss a guide to proceed genetic tests for their efficiency, accessibility and quality in laboratory.

[New medical education of laboratory medicine at Tohoku University].

The purpose of this paper is to give an insight into the medical education program of our division at Tohoku University. Laboratory medicine in medical education is a field of learned basic clinical tests. Students have to learn the clinical laboratory through early clinical exposure in the first grade and try clinical technology through small-group learning in the fifth grade. Finally, they learn laboratory medicine such as infection control in our or another clinical hospital. The objects of our course are to encourage and promote the highest standards of training and post-graduate education of physicians and scientists at our university.

[Laboratory testing for rheumatic diseases].

The diagnosis of rheumatic diseases is primarily based on clinical symptoms and laboratory findings. However, diagnosis of rheumatic disease is often difficult because of the variations even in the same disease. Routine laboratory tests are valuable in detecting renal dysfunctions. In this review, the important auto-antibodies and inflammatory markers associated with rheumatic diseases are described. Further, their utility as diagnostic and prognostic tools, including their specificity, sensitivity and practical applications, is discussed.

[Relationship of polymorphism in CYP2C9 to genetic susceptibility to diclofenac-induced influenza-virus-associated encephalopathy].

The mechanism causing influenza-virus-associated encephalopathy is unclear, even though diclofenac metabolites may induce this pathogenesis. CYP2C9 is known as the major cytochrome P450 gene product that catalyzes diclofenac in human liver. It is uncertain whether the mutation of CYP2C9 is associated the pharmacologic effects of diclofenac in influenza infection. Therefore, we applied a simple and rapid procedure involving real-time fluorescence allele-specific PCR(TaqMan-ASA) assay and denaturing HPLC assay to detect the mutation of CYP2C9 gene. A single-base mutation in the CYP2C9 gene was found in one of thirty subjects in the healthy population. We suggest that this mutation in the CYP2C9 gene may be related to diclofenac-induced influenza-virus-associated encephalopathy.