Hayato Miyachi

FLT3-ITD induces ara-C resistance in myeloid leukemic cells through the repression of the ENT1 expression

Fms-related tyrosine kinase 3-internal tandem duplications (FLT3-ITD) are strongly associated with the refractory nature of acute myeloid leukemia (AML) by the standard combined chemotherapy. FLT3-ITD-expressing murine and human myeloid cell lines, HF6/FLT3-ITD and K562/FLT3-ITD cells, respectively, were developed in order to clarify whether FLT3-ITD is involved in the resistance to cytotoxic agents in AML. Both of these cell lines were specifically resistant to the pyrimidine analogue cytosine arabinoside (ara-C), an essential agent for AML, accompanied by the downregulation of equilibrative nucleoside transporter 1 (ENT1), a transporter responsible for the cellular uptake of ara-C. The ENT1 promoter activity and the cellular uptake of ara-C were reduced in K562/FLT3-ITD cells, and rescued by pretreating the cells with PKC412, a FLT3 inhibitor. In addition, the expression of hypoxia inducible factor 1 alpha subunit (HIF1A) transcripts was upregulated in K562/FLT3-ITD cells, and the induction of HIF-1alpha reduced the promoter activity of ENT1 gene in K562 cells. Taken together, these findings suggest that FLT3-ITD specifically induces ara-C resistance in leukemic cells by the repression of ENT1 expression, possibly through the upregulation of HIF-1alpha, while also partially accounting for the poor prognosis of AML with FLT3-ITD due to resistance to the standard chemotherapy protocols which include ara-C.

Establishment of a xenograft model of human myelodysplastic syndromes.

Background To understand how myelodysplastic syndrome cells evolve from normal stem cells and gain competitive advantages over normal hematopoiesis, we established a murine xenograft model harboring bone marrow cells from patients with myelodysplastic syndromes or acute myeloid leukemia with myelodysplasia-related changes. Design and Methods Bone marrow CD34+ cells obtained from patients were injected, with or without human mesenchymal stem cells, into the bone marrow of non-obese diabetic/severe combined immunodeficient/IL2Rγnull hosts. Engraftment and differentiation of cells derived from the patients were investigated by flow cytometry and immunohistochemical analysis. Results Co-injection of patients’ cells and human mesenchymal stem cells led to successful engraftment of patient-derived cells that maintained the immunophenotypes and genomic abnormalities of the original patients. Myelodysplastic syndrome-originated clones differentiated into mature neutrophils, megakaryocytes, and erythroblasts. Two of the samples derived from patients with acute myeloid leukemia with myelodysplasia-related changes were able to sustain neoplastic growth into the next generation while these cells had limited differentiation ability in the murine host. The hematopoiesis of mice engrafted with patients’ cells was significantly suppressed even when human cells accounted for less than 1% of total marrow mononuclear cells. Histological studies revealed invasion of the endosteal surface by patient-derived CD34+ cells and disruption of extracellular matrix architecture, which probably caused inhibition of murine hematopoiesis. Conclusions We established murine models of human myelodysplastic syndromes using cells obtained from patients: the presence of neoplastic cells was associated with the suppression of normal host hematopoiesis. The efficiency of engraftment was related to the presence of an abnormality in chromosome 7.

A Comparative Study of the Bactericidal Activity and Daily Disinfection Housekeeping Surfaces by a New Portable Pulsed UV Radiation Device

Daily cleaning and disinfecting of non-critical surfaces in the patient-care areas are known to reduce the occurrence of health care-associated infections. However, the conventional means for decontamination of housekeeping surfaces of sites of frequent hand contact such as manual disinfection using ethanol wipes are laborious and time-consuming in daily practice. This study evaluated a newly developed portable pulsed ultraviolet (UV) radiation device for its bactericidal activity in comparison with continuous UV-C, and investigated its effect on the labor burden when implemented in a hospital ward. Pseudomonas aeruginosa, Multidrug-resistant P. aeruginosa, Escherichia coli, Acinetobacter baumannii, Amikacin and Ciprofloxacin-resistant A. baumannii, Staphylococcus aureus, Methicillin-resistant S. aureus and Bacillus cereus were irradiated with pulsed UV or continuous UV-C. Pulsed UV and continuous UV-C required 5 and 30xa0s of irradiation, respectively, to attain bactericidal activity with more than 2Log growth inhibition of all the species. The use of pulsed UV in daily disinfection of housekeeping surfaces reduced the working hours by half in comparison to manual disinfection using ethanol wipes. The new portable pulsed UV radiation device was proven to have a bactericidal activity against critical nosocomial bacteria, including antimicrobial-resistant bacteria after short irradiation, and was thus found to be practical as a method for disinfecting housekeeping surfaces and decreasing the labor burden.

Sonographic Appearance of the Submandibular Glands in Patients With Immunoglobulin G4-Related Disease

Swelling of the salivary glands is often an initial sign of immunoglobulin G4 (IgG4)‐related disease or IgG4‐related sclerosing/autoimmune disease. We encountered 2 patients with IgG4‐related disease who showed swollen submandibular glands with a unique characteristic sonographic pattern. Bilateral submandibular glands of both patients were enlarged with a smooth contour. The internal echo texture indicated multiple hypoechoic foci scattered against a heterogeneous background, which characteristically appeared with a mottled or irregular netlike appearance. A histopathologic examination of a resected section showed multiple foci of dense infiltrated lymphoplasmacytic cells and lymph follicles encircled by fibrous bands. A mottled appearance in the sonographic findings of the submandibular glands suggests the characteristic of IgG4‐related disease and can be helpful in the differential diagnosis at the initial manifestation.

The tortoiseshell pattern in one or both sides of the submandibular glands in mucosa-associated lymphoid tissue lymphoma is related to chromosomal aberrations and the disease extent.

Objective. Lesions of mucosa‐associated lymphoid tissue (MALT) lymphoma in the submandibular glands are localized or a part of systemic involvement in association with chromosomal aberrations. This series was undertaken to investigate the sonographic features of MALT lymphoma in the submandibular glands and their relationships with chromosomal aberrations and the disease extent. Methods. A total of 5 patients with MALT lymphoma without Sjögren syndrome in the submandibular glands were enrolled in this series. Patients underwent sonography of the submandibular glands with a high‐resolution transducer before surgical biopsy of the main lesion. Sonographic characteristics of the lesions were described for their location, presence of a posterior echo, texture, and presence of an internal echo. Results. Sonography in all cases showed hypoechoic and solid masses with increased posterior echo enhancement. There was an arrangement of hypoechoic small compartments demarcated by hyperechoic contour lines, which had a tortoiseshell pattern. This pattern was classified into 2 types according to its location: a lesion in the right or left side and lesions in both sides of the submandibular glands, found in 3 and 2 patients, respectively. The latter 2 cases had chromosomal aberrations of t(11;18)(q23;q23) and t(12;18)(q22;q21), respectively, and were revealed as secondary organ involvement. Conclusions. The sonographic appearance of MALT lymphoma in the submandibular glands was characterized by the tortoiseshell pattern in both primary and secondary lesions. Detection of this pattern in both sides of the submandibular glands can be an indicator of chromosomal aberrations and systematic involvement of the disease.

Genome Sequences of Multidrug-Resistant Acinetobacter baumannii Strains from Nosocomial Outbreaks in Japan

ABSTRACT Acinetobacter baumannii has emerged worldwide as an important nosocomial pathogen in medical institutions. Here, we present the draft genome sequences of A. baumannii strains MRY09-0642, MRY10-0558, and MRY12-0277 that were isolated from nosocomial outbreaks in Japan between 2008 and 2012 and that are resistant to antimicrobial agents, including carbapenems, fluoroquinolones, and aminoglycosides.

Clinical and pathological features of B-cell non-Hodgkin lymphomas lacking the surface expression of immunoglobulin light chains.

Abstract Background: The flow cytometric analysis of surface immunoglobulin light chains (sIgL) is used as a simple method for evaluating monoclonal B-cell proliferation. However, the sIgL expression, κ or λ, is occasionally undetectable in cases with B-cell non-Hodgkin lymphomas (B-NHL). The purpose of this study was to investigate the clinical and pathological characteristics of these B-NHL cases. Methods: We retrospectively analyzed 50 cases with previously untreated sIgL-negative B-NHL. All of these cases had been diagnosed at Tokai University Hospital between January 2001 and February 2011. Their medical charts were reviewed. Results: These cases had several clinical features: diffuse large B-cell lymphoma (DLBCL) (72%), a high serum lactate dehydrogenase level (66%), clinical stage III and IV (68%), and complex karyotypes (58%). Seven out of eight evaluated patients (87%) did not express cytoplasmic IgL, and the DNA rearrangement pattern of IgL showed diversity in 10 analyzed patients. The 5-year event-free survival of all the sIgL-negative B-NHL cases was significantly better with rituximab-containing chemotherapies in comparison to the regimens without it (57.9% vs. 17.9%, p=0.0207), although there was no statistical significance when the DLBCL cases were analyzed (56.6% vs. 22.2%, p=0.1530). Conclusions: These findings suggest that sIgL-negative B-NHL cases predominantly developed DLBCL in advanced disease, but were heterogeneous at the molecular level.

Differential Coexpression of Mex Efflux Pumps in a Clinical Strain of Metallo-β-lactamase-producing pseudomonas aeruginosa During the Stepwise Evolution of Resistance to Aminoglycosides

Background: We observed a stepwise evolution of resistance to aminoglycosides in Pseudomonas aeruginosa by tracking the development of multidrug resistance in a series of clinical isolates from a patient with a serious burn injury. Methods and Results: Fifteen isolates sequentially recovered from burn wounds fell into 4 groups (group 1 [G1], G2, G3, and G4) in the order of appearance in the evolution of resistance as assessed by antibiotic susceptibility profiles. In contrast to G1, which was sensitive to aminoglycosides, there was a stepwise evolution in resistance to aminoglycosides: gentamicin alone (G2), gentamicin and tobramycin (G3), and finally, amikacin (a panaminoglycoside/multidrug resistance; G4). All but the G1 strains were positive for metallo-&bgr;-lactamase and resistant to carbapenems and fluoroquinolones. When the isolates were screened for the expression of Mex efflux pumps by reverse transcriptase polymerase chain reaction, all 4 groups showed expression of the mexA but not mexC genes. The expression of mexE messenger RNA was observed in G2, G3, and G4, and that of mexX was observed in G4. The first isolates recovered for each of the 4 groups were further characterized as their representatives (G1-1, G2-1, G3-1, and G4-1) for the expression levels of Mex efflux pumps by a Western blotting analysis. In concomitance with the evolution of drug resistance, MexE protein was found to be differentially expressed. It was expressed in G2-1, up in G3-1 and back down in G4-1. The MexX protein was expressed only in G4-1 and may play a role in establishing amikacin resistance. Conclusions: These results suggest that a series of clinical isolates of a metallo-&bgr;-lactamase-producing P. aeruginosa has acquired panaminoglycoside-resistant profiles in a stepwise manner via differential coexpression of the Mex efflux pumps.

A novel aberrant form of e13a2 BCR-ABL1 transcript in chronic myelogenous leukemia undetectable with the standardized real-time quantitative polymerase chain reaction from the Europe Against Cancer Program.

The detection and monitoring of BCR-ABL1 transcripts using real-time quantitative polymerase chain reaction (RQ-PCR) is indispensable for the management of patients with chronic myelogenous leukemia (CML) (1). The Europe Against Cancer (EAC) Program has established a standardized RQ-PCR protocol for BCR-ABL1 assay reproducibility and data comparison among laboratories (2, 3). The RQ-PCR primer set, ENF501 and ENR561 (Figure 1A), is designed to amplify the two major types of BCR-ABL1 transcripts, e13a2 and e14a2, where either BCR exon 13 (e13) or 14 (e14) is fused to ABL exon 2 (a2), respectively. We recently experienced a CML patient who harbored a novel form of aberrant e13a2 transcript, which was undetectable with the EAC protocol. A 62-year-old Japanese woman was diagnosed with CML based on the detection of t(9;22)(q34;q11.2). Fluorescent in situ hybridization (FISH) analysis revealed that 92.8% of bone marrow cells were positive for BCR-ABL1. SYBR Green I RQ-PCR with the EAC primer set, however, could not detect the BCR-

Elimination of interference by lipids in the low WBC mode in the automated hematology analyzer XN-2000.

Sir, The dissemination of chemotherapy in various malignancies and expanded application of stem cell transplantation have currently made more chances for the physicians to take care of the patients with leukopenia [1, 2]. There is a demand for immediate and reliable data for the low counts of white blood cells (WBC) in automated hematology analyzers. However, the measurement of WBC counts and differentials has a limitation in the accuracy in leukopenia. Particularly spuriously high counts by the contamination of nucleated erythrocytes (NRBC) and lipids are problematic in the care of the leucopenia patients [3]. Conventional analyzers usually use scattered lights or direct-current electricity to count WBC, and thus, the intensities are subject to interference from such a non-WBC particle. The newly developed automated hematology analyzers XN-series (Sysmex Corp., Kobe, Japan) have several modifications for the reliability of measurement by optimization of the reagent reactions, the signal processing, and the analysis algorithms: The contamination of NRBC in the measurement of WBC counts is eliminated using WNR channel, and the accurate WBC differentials are provided using WDF channel [4]. Using fluorescence staining for nucleic acid and scattered light, both measurement channels exclude the interference such as lipid, nonlysed RBCs, and so on. Furthermore, using the WDF channel, the XN-series are also equipped with the low WBC (LW) mode, which is intended for measurement of a low range of WBC count and differentiation by counting threefold sample volume [4]. We have evaluated the assay performance of WBC counting of XN-2000 in a low range by comparing the LW and the normal measurement mode (Whole blood mode: WB mode), the conventional analyzer XE-2100 (Sysmex Corp.), and the manual method. Clinical samples used in the study were submitted to the clinical laboratory of Tokai University Hospital for a complete blood count test and sampled during a 6-month period from September 2010 to March 2011. Peripheral blood was taken with addition of EDTA-2K as an anticoagulant. The study was approved by Institutional Review Board for Clinical Research of Tokai University Hospital (12R116) and Sysmex Corp. When the within-run reproducibility in 10 samples with a LW count (<1.40 9 10/L, three of them were <0.13 9 10/L) was determined in 5 or 10 replicates, the LW mode had better within-run reproducibility of WBC counting than the WB mode, with the coefficients of variation (CV%) being 0.6–7.7% and 1.6–11.2%, respectively. The LW mode also provided better within-run reproducibility than the WB mode in the analysis of the absolute numbers of each cell fraction of WBC in neutrophil and lymphocyte, with the CV being <15% in the LW mode, as long as the absolute count of each differential fraction was more than 0.10 9 10/L (data not shown). This improvement in the measurement reproducibility in LW mode is confirmatory of WBC counting using threefold more cells in the samples in comparison with the WB mode. The LW mode showed the linearity of WBC counting in a range 0.01–1.15 9 10/L (y = 0.990x + 0.002, r = 0.9997), when evaluated with an eleven-step dilution series using the diluent CELLPACK (Sysmex Corp.). The linearity was also seen in each WBC differential measured by the LW and WB modes: 0.01– 0.33 9 10 and 0.02–0.33 9 10/L for neutrophils, 0.01– 0.61 9 10 and 0.05–0.56 9 10/L for lymphocytes. Then, we evaluated the interference of lipid particles for WBC counting by the addition of fat emulsion to a sample from a healthy volunteer. XN-2000 WNR and WDF channel for WB and LW mode could accurately count WBC by distinguishing it from the lipids (Figure 1a and b). On the other hand, XE-2100 showed false elevation of WBC counts, with a curved strand of ‘Lissajous-like pattern’ plots in the scattergram (Figure 1b) [5, 6]. The method comparison for WBC counting between the LW and WB modes in XN-2000 and XE-2100 showed good correlation in the range of <1.40 9 10/L (293, 298 and 294 samples, respectively; Figure 2a). However, 8 of the samples showed discrepancy in the