Infective Endocarditis Caused by Panton-Valentine Leukocidin-producing Methicillin-susceptible Staphylococcus aureus Identified by the Broad-range PCR Method.

A 76-year-old man was admitted to a community hospital due to a persistent high fever. He became afebrile after the administration of broad-spectrum antibiotics, but developed heart failure due to progressive aortic and mitral valve insufficiency and was transferred to our hospital. Although sequential blood cultures were negative, a broad-range polymerase chain reaction targeting the bacterial 16S-rRNA gene followed by the direct sequencing of whole blood revealed spa(+), mecA(-) and Panton-Valentine leukocidin (PVL)(+). He was finally diagnosed with infective endocarditis (IE) caused by PVL-producing methicillin-susceptible Staphylococcus aureus (MSSA), and underwent cardiac surgery. This is the first reported case of IE due to MSSA producing PVL.

[Issues of Personalized Medicine from the Viewpoint of Laboratory Medicine].

Personalized medicine is expected to provide patients with safe and effective treatment compared to conventional medical care in which patients are treated based on the diagnosis and/or histology. In personalized medicine, patients are treated based on their genetic makeup and genetic characteristics of tumor tissues in the case of cancer chemotherapy. Genomic biomarker tests are used to molecularly characterize host and tumor tissues and stratify patients for the appropriate drugs. Drugs targeting the causative genetic changes have been developed along with companion diagnostics to test such genetic changes. In this paper, I introduce the technical guidance for companion diagnostics and related drugs issued by the Pharmaceutical and Medical Devices Agency of Japan, and discuss how to carry out a concordance study of diagnostic tests for the ALK fusion gene when new ALK inhibitors are approved. The regulations for companion diagnostics should be revised frequently to keep up with advances in this area.

[Trends and Challenges of the Statutory Regulation of Molecular Genetic Tests].

Advances in basic science have made it possible to characterize tumors at molecular levels and exploit the differences in the genetic makeup of tumors for personalized cancer treatment. Biomarker tests are essential to stratify patients with the same tumor histology for appropriate treatment. Such tests are developed together with drugs, involving mainly molecular genetic tests at present, and are called companion diagnostics (CDx). Under the universal health care system in Japan, molecular genetic tests are permitted in clinics, and the fees are reimbursed only when approved diagnostic reagents or kits are used. However, new tests are developed so fast that the regulation cannot keep up with the pace. To fill this gap, the framework of advanced medical technologies was introduced in 1984. In 2012, this framework was amended to classify medical technology using unapproved diagnostics or home-brew assays as advanced medical technologies A. In my talk at this symposium, trends and challenges of the statutory regulation of molecular genetic tests in Japan were discussed, followed by personal proposals to advance the clinical application of novel medical technologies in the field of personalized cancer treatment.

A novel FOXP1-PDGFRA fusion gene in myeloproliferative neoplasm with eosinophilia.

We identified a novel fusion gene, FOXP1-PDGFRA, in a patient with myeloproliferative neoplasm (MPN) with eosinophilia, harboring the chromosome abnormality t(3;4)(p13;q12). The patient responded well to imatinib and has remained in molecular remission for 3 years. This is the seventh fusion gene involving PDGFRA in MPN with eosinophilia. PDGFRA was truncated in its autoinhibitory domain, as in other PDGFRA-related MPNs, and was fused to FOXP1 at its functional forkhead domain. Comparing genomic DNA with mRNA sequences provides the possibility that the splicing process near the breakpoint junction in the FOXP1-PDGFRA fusion gene may use the normal splice donor site for intron 23a of FOXP1 and the cryptic splice acceptor site in exon 12 of PDGFRA. This is the first report to describe the FOXP1-PDGFRA fusion gene in MPN.

Substitution in Amino Acid 70 of Hepatitis C Virus Core Protein Changes the Adipokine Profile via Toll-Like Receptor 2/4 Signaling.

BACKGROUND & AIMS: It has been suggested that amino acid (aa) substitution at position 70 from arginine (70R) to glutamine (70Q) in the genotype 1b hepatitis C virus (HCV) core protein is associated with insulin resistance and worse prognosis. However, the precise mechanism is still unclear. The aim of this study was to investigate the impact of the substitution at position 70 in HCV core protein on adipokine production by murine and human adipocytes. METHODS: The influence of treatment with HCV core protein (70R or 70Q) on adipokine production by both 3T3-L1 and human adipocytes were examined with real-time PCR and enzyme-linked immunosorbent assay (ELISA), and triglyceride content was also analyzed. The effects of toll-like receptor (TLR)2/4 inhibition on IL-6 production by 3T3-L1 induced by HCV core protein were examined. RESULTS: IL-6 production was significantly increased and adiponectin production was reduced without a change in triglyceride content by treatment with 70Q compared to 70R core protein in both murine and human adipocytes. IL-6 induction of 3T3-L1 cells treated by 70Q HCV core protein was significantly inhibited with anti-TLR2 antibody by 42%, and by TLR4 inhibitor by 40%. CONCLUSIONS: Our study suggests that extracellular HCV core protein with substitution at position 70 enhanced IL-6 production and reduced adiponectin production from visceral adipose tissue, which can cause insulin resistance, hepatic steatosis, and ultimately development of HCC.

Corrigendum: Human ß-defensin-3 inhibits migration of colon cancer cells via downregulation of metastasis-associated 1 family, member 2 expression.

In this article, Fig. 2 is incorrect. The corrected Fig. 2 is shown using data from the tissue array samples. The new figure demonstrates the same findings as the original figure. Accordingly, in the paragraph of Materials and methods, the sentence '...surgically resected colon cancer tissues' and 'This study was...research committee' and in the paragraph of Results, the sentence 'Similar results were...tissue array samples' should be deleted. The above changes do not alter the original conclusions of this study. [the original article was published in the International Journal of Oncology 45: 1059-1064, 2014 DOI: 10.3892/ijo.2014.2507].

Effect of Genetic Polymorphism of CYP3A5 and CYP2C19 and Concomitant Use of Voriconazole on Blood Tacrolimus Concentration in Patients Receiving Hematopoietic Stem Cell Transplantation.

BACKGROUND: Blood tacrolimus (TAC) concentration delivered via intravenous administration is known to be influenced by genetic polymorphism of CYP3A5 and interaction with triazole antifungal agents. However, interindividual variability of blood TAC concentration is as of yet still difficult to predict during the early stages of hematopoietic stem cell transplantation (HSCT). This study was conducted to assess the wide variability of blood TAC concentrations because of the hepatic metabolic activities of CYP3A and CYP2C19 in HSCT recipients. METHODS: This study is a single-institute prospective study that includes 21 adult patients who underwent HSCT and received 24 hours continuous intravenous administration of TAC at the Mie University Hospital between January 2009 and March 2014. After HSCT, the changes in blood TAC concentration/dose (C/D) ratio and TAC dose reduction from initial dose were investigated. RESULTS: Significant differences between HSCT recipients with CYP3A5*1 allele and CYP3A5*3/*3 genotype were observed with respect to the median TAC C/D ratio on day 14 (563 versus 742 ng/mL per mg/kg, P < 0.01) and day 21 (672 versus 777 ng/mL per mg/kg, P < 0.05) after HSCT. Concomitant administration of voriconazole (VRCZ), but not of lansoprazole, was found to significantly increase the median TAC C/D ratio on day 14 (557 versus 723 ng/mL per mg/kg, P < 0.01). Possession of the CYP3A5*3/*3 genotype (day 14: odds ratio, 32.2; day 21: odds ratio, 33.0; P < 0.05) and concomitant administration of VRCZ (day 14: odds ratio, 37.8; P < 0.05) were found to be independent risk factors, which significantly contributed to an increased TAC C/D ratio. In HSCT recipients with CYP3A5*3/*3 genotype (78.0%), the median TAC dose ratio (day 21/day -1) was significantly lower compared with HSCT recipients with the CYP3A5*1 allele (94.1%), whereas VRCZ administration itself had no significant influence. Interestingly, in HSCT recipients with CYP2C19*1/*1, we found that the influence of VRCZ on the TAC dose ratio (85.7%) was relatively mild, even in a recipient with CYP3A5*3/*3. CONCLUSIONS: In HSCT recipients, the variability of intravenous TAC concentration in the blood could be explained in part by the genetic variation of CYP3A5. The study results also strongly imply that the magnitude of hepatic interaction between TAC and VRCZ is affected by the genetic polymorphism of both CYP3A5 and CYP2C19 genes.

Human ß-defensin-3 inhibits migration of colon cancer cells via downregulation of metastasis-associated 1 family, member 2 expression.

The innate immune system plays an important role as the first line of defense against many types of microbes. Accumulating reports suggest that human ß-defensins (hBDs) are expressed by and have certain roles in some cancer cells. In this study, we investigated the roles of hBD-3 in colon cancer cells. The expression of hBD-3 was examined by reverse transcriptase-polymerase chain reaction analysis of colon cancer cell lines and immunohistochemical staining of colon cancer tissues. The effect of hBD-3 on proliferation of colon cancer was assessed using the MTT assay and a real-time cell analyzer, and the effect of hBD-3 on the migration of colon cancer cells was also examined. The results showed that hBD-3 is not expressed in colon cancer cells but is produced by tumor-infiltrating monocytes. Migration of colon cancer cells was significantly inhibited by hBD-3 in a dose-dependent manner, although proliferation of colon cancer cells was not affected by administration of hBD-3. Moreover, reduced expression of metastasis-associated 1 family, member 2 (MTA2) mRNA in colon cancer cells was associated with exposure to hBD-3. In conclusion, progression of colon cancer was inhibited by hBD-3 in a paracrine fashion. Therefore, hBD-3 may be a potent new agent for treating colon cancer.

Evaluation of clinical performance of the major BCR-ABL mRNA detection kit which enables conversion to international standard scale using the reference material calibrator.

In a multicenter study, we evaluated the Major BCR-ABL mRNA/ABL mRNA quantification kit (M135R), which uses reference material included in the kit designed to report results using the international scale (IS). In total, 127 samples were studied. A good correlation was observed between M135R results and home-brew RT-qPCR results, which are reported on the IS using a conversion factor (r=0.90; n=115). However, the correlation coefficient between M135R results and Amp-CML results was relatively low (r=0.56; n=108). A good correlation was observed between M135R results from the two assay sites (r=0.94; n=115). The subset analysis of samples from the two assay sites showed M135R to have a good correlation even in the low IS range (r=0.98; IS≤1%). M135R showed high sensitivity and accuracy for detecting minimal residual disease and is considered to be a useful tool for treatment response assessment and for early detection of recurrence in CML patients.

Resveratrol sensitizes HepG2 cells to TRAIL-induced apoptosis.

Resveratrol is a natural polyphenol found in a wide variety of plants, including grapes, berries, and peanuts. Resveratrol can modulate a wide spectrum of molecular targets, including those involved in cancer signaling pathways. Here, we evaluated the role of resveratrol in tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and examined the molecular mechanisms in the human hepatocellular carcinoma cell line HepG2. We used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay to assess cell viability, flow cytometry to analyze cell cycle and apoptosis, and immunoblotting to detect protein expression. Resveratrol decreased cell viability at a concentration of 100 µmol/l or higher. At a concentration of 50 µmol/l, resveratrol induced S phase arrest of the cell cycle without apoptosis. In addition, phospho-AMPK increased significantly in a dose-dependent manner. Resveratrol was found to synergistically augment TRAIL-induced apoptosis. The rates of early apoptosis were 3.4, 9.6, and 49.6% on treatment with 50 µmol/l resveratrol, 10 ng/ml TRAIL, and both reagents, respectively. Resveratrol significantly downregulated the expression of survivin in a dose-dependent manner. In conclusion, we found that that resveratrol could augment TRAIL sensitivity by downregulating survivin. These results suggest that combination resveratrol with TRAIL may be an effective new strategy for the treatment of hepatocellular carcinoma.

Collagen triple helix repeat containing 1 is overexpressed in hepatocellular carcinoma and promotes cell proliferation and motility.

Although several therapeutic options are available for hepatocellular carcinoma (HCC), the outcome is still very poor. One reason is the complexity of signal transduction in the pathogenesis of HCC. The aim of this study was to identify new HCC-related genes and to investigate the functions of these genes in the pathogenesis and progression of HCC. Whole genomes of 15 surgically resected HCC specimens were examined for copy number alterations with comparative genomic hybridization. Gene expression was compared between HCC and normal liver tissues. The roles of the new genes in the progression of HCC were studied using cultured cell lines. Copy number gain in chromosome 8q was detected in 53% of HCC tissues examined. The gene that coded for collagen triple helix repeat containing 1 (CTHRC1), located at chromosome 8q22.3, was overexpressed in HCC compared with normal or liver cirrhosis tissues and identified as a new HCC-related gene. CTHRC1 deletion with short hairpin RNA significantly reduced proliferation, migration and invasion of HepG2 and Huh7 cells. In addition, mRNA of integrins ß-2 and ß-3 was downregulated, with deletion of CTHRC1 in these cells. Immunohistochemical staining on resected HCC tissues showing positive staining areas for CTHRC1 was significantly greater in poorly-differentiated HCC compared with well­differentiated HCC. Moreover, some cases showed strong staining for CTHRC1 in invasive areas of HCC. CTHRC1 has the potential to be a new biomarker for the aggressive HCC, and to be a new therapeutic target in treating HCC.

Prolonged thrombocytopenia after living donor liver transplantation is a strong prognostic predictor irrespective of splenectomy: the significance of ADAMTS13 and graft function [corrected].

The precise mechanism of prolonged thrombocytopenia following living donor liver transplantation (LDLT) remains unclear. To determine risk factors associated with prolonged thrombocytopenia following LDLT, with a focus on the activity of ADAMTS13 (a disintegrin-like and metalloproteinase with thrombospondin type-1 motifs member 13) and the influence of splenectomy. Adult LDLT patients were divided into two groups on the basis of platelet counts (100 × 10(3)/µL) on POD 14: high and low platelet (HP and LP) groups. Survival analysis was performed in the 100 patients, and ADAMTS13 activity and von Willebrand factor (VWF) levels in the plasma were measured in 65 adult recipients. The 6-month survival rate was significantly lower in the LP group (n = 36) than in the HP group (n = 62) (61.1 vs. 93.5 %). ADAMTS13 activity had been significantly lower in the LP group (n = 23) than in the HP group (n = 42). The VWF/ADAMTS13 ratio was significantly higher in the LP group than in the HP group. The independent risk factors for thrombocytopenia on POD14 were preoperative AT levels and ADAMTS13 activity on POD14. TPO levels on POD14 were significantly higher in the LP group than in the HP group, while those on POD28 in the LP group were significantly decreased, despite the low platelet levels. Irrespective of splenectomy, platelet counts and ADAMTS13 activity in the LP group remained low until POD28, while VWF/ADAMTS13 ratio significantly increased until POD28. These results suggest that prolonged thrombocytopenia after LDLT was associated with not only a decrease in ADAMTS13 due to sinusoidal endothelial cell injury, but also low TPO production due to hepatocyte dysfunction, irrespective of splenectomy [corrected].

Validation of homogeneous assays for HDL-cholesterol using fresh samples from healthy and diseased subjects.

BACKGROUND: High-density lipoprotein-cholesterol (HDL-C) is a negative risk factor for cardiovascular events. Although several homogeneous HDL-C assays are available, their accuracy has not been validated, particularly in subjects with disease. We aimed to clarify whether HDL-C concentrations measured by homogeneous assays [HDL-C (H)] agree with those determined by the reference measurement procedures [HDL-C (RMP)] using ultracentrifugation and precipitation with heparin-manganese reagent in fresh clinical samples. METHODS: HDL-C concentrations in samples from 48 healthy subjects and 119 subjects with disease were determined using 12 homogeneous assays and RMPs. RESULTS: All reagents showed excellent intra- and inter-assay CVs (<2.23%) for two pooled sera. Furthermore, the mean bias was within ± 1.0% in nine reagents using samples from healthy subjects and in eight reagents using samples from subjects with disease. In a single HDL-C (H) determination, the total error requirement of the National Cholesterol Education Program (95% of results < 13%) was fulfilled in nine reagents using samples from healthy subjects and six reagents in those from subjects with disease. Error component analysis revealed that only one reagent exceeded ± 10% total error in samples from healthy subjects, whereas four reagents exceeded this error in samples from subjects with disease. Correlations between HDL-C (H) and HDL-C (RMP) revealed that the slopes were within 1.00 ± 0.06 in six reagents in healthy subjects, and eight reagents in subjects with disease. CONCLUSIONS: Except for three reagents, HDL-C (H) agrees well with HDL-C (RMP) in subjects with common disease, but not in those with extremely low HDL-C or abnormal HDL composition.

[Development of Rapid Assay of Transthyretin in Whole Blood].

Transthyretin (TTR), a rapid turnover protein with a half-life of 2 days, which is shorter than that of albumin, is considered to be a sensitive indicator of a patient's nutritional status. We have newly developed a rapid assay of TTR by immunochromatography (IC) using samples of whole blood as well as serum at volumes of 10 µL. The fundamental performance was evaluated using control sera. In addition, whole blood samples were measured by IC and the results were compared with measured levels of plasma samples by turbidimetry and those of sera by nephelometry. The limit of quantification was 8.3 mg/dL. The linearity was 8.7-35.4 mg/dL. The reproducibility was 6.1-8.6% coefficient of variation. Bilirubin, hemolysis, chyle, and rheumatoid factor did not influence the measured levels. An increase of 1% in hematocrit resulted in a decrease of 1% in the measured levels. The correlation between IC (y) and nephelometry (x) was determined to be y = 0.927x+2.62 (r = 0.935, n = 64). The newly developed IC showed good performance and it was suggested that IC would be useful for nutrition monitoring as point of care test.

Hemodynamic and pathophysiological characteristics of intradialytic blood pressure elevation in patients with end-stage renal disease.

An increase in systolic blood pressure (SBP) after hemodialysis (intradialytic-HTN) is associated with adverse outcomes in patients on regular hemodialysis. However, the hemodynamic and Doppler echocardiographic characteristics of intradialytic-HTN and its impact on clinical outcomes are unclear. A retrospective analysis of 84 patients (45 men, 70±9 years) stratified into three groups on the basis of SBP response from pre- to post-hemodialysis: GHTN (intradialytic-HTN, SBP increase 10 mm Hg), GDROP<15 mm Hg (SBP drop <15 mm Hg), and GDROP15 mm Hg (SBP drop 15 mm Hg). Hemodynamic and echocardiographic assessments were performed pre- and post-hemodialysis, and patients were followed for 41±17 months. GHTN had higher blood glucose and lower baseline SBP, serum potassium and total cholesterol. Cardiothoracic ratio was smaller, and peak early diastolic mitral annular velocity (E') was lower in GHTN. During hemodialysis, SBP and diastolic blood pressure increased only in GHTN. After hemodialysis, left ventricular (LV) filling pressure (E/E' ratio) decreased only in GDROP15 mm Hg, resulting in a higher E/E' ratio in GHTN than GDROP15 mm Hg. Multivariate logistic regression analysis revealed a positive correlation between blood glucose and intradialytic-HTN, whereas cardiothoracic ratio, pre-hemodialysis SBP and the change in E/E' ratio with hemodialysis were negatively related to intradialytic-HTN. During follow-up, GHTN had more cardiovascular deaths than GDROP15 mm Hg (P=0.03). Multivariate Cox regression analysis showed that lower serum potassium and previous coronary artery disease, but not intradialytic-HTN, were associated with cardiovascular deaths. A higher LV afterload and elevated filling pressures after hemodialysis, indicative of increased cardiovascular stiffening and impaired diastolic filling, may contribute in part to an increased cardiovascular burden in patients with intradialytic-HTN.

[Discussing the usefulness of companion diagnostics - introductory remarks by chairpersons].

Personalized medicine has been expected to be utilized in daily practice since the international human genome project completed sequencing of the entire genome in 2003. The aim of personalized medicine is to treat patients effectively and safely based on the genome characteristics of each patient, or to choose the right drug for the right patient at the right time and at the right dose. Regulatory agencies such as the United States Food and Drug Administration(FDA) and Pharmaceuticals and Medical Devices Agency (PMDA), Japan implemented guidance to facilitate the co-development of drugs and companion diagnostics. For the practice of personalized medicine, companion diagnostic tests are essential, although their availability is limited.

[Potential and perspective of genetic testing in Renaissance of laboratory medicine].

Among molecular genetic tests, tests for genomic biomarkers are increasingly performed in order to decide the right drug for the right patient at the right time. These genetic tests, including pharmacogenomics (PGx) tests, are essential for personalized medicine. Companion diagnostics (CDx) will be co-developed from the early stage of drug development and used for patient stratification in Phases II/III of clinical trials. The strategic change in development from blockbuster drugs to molecularly targeted drugs will enhance the clinical application of genetic tests, including CDx and PGx. The genetic tests utilized for making treatment decisions have the potential to regenerate laboratory medicine.