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FMS-like tyrosine kinase 3 (FLT3) internal tandem duplication (ITD) mutations in acute myeloid leukemia (AML) are associated with poor prognosis and therapy resistance. This study aimed to demonstrate that inhibiting the deubiquitinating enzymes ubiquitin-specific peptidase 14 (USP14) and ubiquitin C-terminal hydrolase L5 (UCHL5) (USP14/UCHL5) with b-AP15 or the organogold compound auranofin (AUR) induces apoptosis in the ITD-transformed human leukemia cell line MV4-11 and mononuclear leukocytes derived from patients with FLT3-ITD-positive AML. This study included patients diagnosed with AML at Tokyo Medical and Dental University Hospital between January 2018 and July 2024. Both treatments blocked downstream FLT3 pathway events, with the effects potentiated by USP14 knockdown. Both treatments inhibited FLT3 deubiquitination via K48 and disrupted translation initiation via 4EBP1, a downstream FLT3 target. FLT3 was downregulated in the leukemic cells, with the associated activation of stress-related MAP kinase pathways and increased NF-E2-related factor 2. Furthermore, the overexpression of B-cell lymphoma-extra-large and myeloid cell leukemia-1 prevented the cell death caused by b-AP15 and AUR. These results suggest that inhibiting USP14/UCHL5, which involves multiple regulatory mechanisms, is a promising target for novel therapies for treatment-resistant FLT3-ITD-positive AML.

Although it is known that BK polyomavirus (BKPyV) causes hemorrhagic cystitis (HC) after allogeneic hematopoietic stem cell transplantation (HSCT), the clinical significance of BKPyV viremia has not been fully evaluated. We retrospectively analyzed the results of quantitative polymerase chain reaction (PCR) evaluations for detecting BKPyV in the whole blood samples of patients undergoing allogeneic HSCT during the period from January 2010 to June 2020 at a single institute, Tokyo Medical and Dental University. BKPyV was detected in the blood of 28 of the 107 evaluated patients, and the cumulative incidence of was 27.9% (95%CI: 20.2-37.9%). HC due to BKPyV developed in four of the 28 patients with BKPyV viremia (14.3%) and in two of the 79 patients without it (2.5%; P < 0.05). BKPyV viremia itself did not affect the patients' post-transplant estimated glomerular filtration rate (eGFR), but BKPyV viremia with a high viral load was significantly associated with decreased eGFR values (P < 0.05). BKPyV viremia was also associated with significantly lower progression-free survival at 3 years (35.1% [95%CI: 17.8-53.1%] vs. 60.4% [95%CI: 48.4-70.5], P < 0.05). Our findings demonstrated that BKPyV viremia was associated with onset of HC, an early decline of renal function, and poorer survival after allogeneic HSCT. Further studies are needed to test these results and elucidate the mechanisms of renal dysfunction associated with BKPyV viremia., (© 2024. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Coronavirus disease (COVID-19) has caused extensive mortality globally; therefore, biomarkers predicting the severity and prognosis of COVID-19 are essential. This study aimed to evaluate the application of presepsin (P-SEP) and thrombomodulin (TM), which are biomarkers of sepsis and endothelial dysfunction, respectively, in the prognosis of COVID-19. Serum P-SEP and TM levels from COVID-19 patients (n = 183) were measured. Disease severity was classified as mild, moderate I, moderate II, or severe based on hemoglobin oxygen saturation and the history of intensive care unit transfer or use of ventilation at admission. Patients in the severe group were further divided into survivors and non-survivors. P-SEP and TM levels were significantly higher in the severe group than those in the mild group, even after adjusting for creatinine values. In addition, TM levels were significantly higher in non-survivors than in survivors. Changes in the P-SEP levels at two time points with an interval of 4.1 ± 2.2 days were significantly different between the survivors and non-survivors. In conclusion, TM and continuous P-SEP measurements may be useful for predicting mortality in patients with COVID-19. Moreover, our data indicate that P-SEP and TM values after creatinine adjustment could be independent predictive markers, apart from renal function., (© 2023. The Author(s).)

Background: Thrombosis is a unique complication of coronavirus disease 2019 (COVID-19). Although antiphospholipid antibodies (aPL) are detected in COVID-19 patients, their clinical significance remains elusive. We evaluated the prevalence of aPL and serum concentrations of beta-2 glycoprotein I (β2GPI), a major self-antigen for aPL, in Japanese COVID-19 patients with and without thrombosis., Methods: This retrospective single-center nested case-control study included 594 hospitalized patients with COVID-19 between January 2020 and August 2021. Thrombotic complications were collected from medical records. Propensity score-matching method (PSM) (1:2 matching including age, sex, severity on admission, and prior history of thrombosis) was performed to compare the prevalence and titer of aPL (anti-cardiolipin (aCL) IgG/IgM, anti-β2GPI IgG/IgM/IgA, and anti-phosphatidylserine/prothrombin antibody (aPS/PT) IgG/IgM) and serum β2GPI concentration. In addition, PSM (1:1 matching including age and sex) was performed to compare the serum β2GPI concentration between COVID-19 patients and healthy donors., Results: Among the patients, 31 patients with thrombosis and 62 patients without were compared. The prevalence of any aPLs was indifferent regardless of the thrombosis (41.9% in those with thrombosis vs. 38.7% in those without, p =0.82). The positive rates of individual aPL were as follows: anti-CL IgG (9.7% vs. 1.6%, p =0.11)/IgM (0% vs. 3.2%, p =0.55), anti-β2GP1 IgG (22.6% vs. 9.7%, p =0.12)/IgA (9.7% vs. 9.7%, p =1.0)/IgM (0% vs. 0%, p =1.0), and anti-PS/PT IgG (0% vs. 1.6%, p =1.0)/IgM (12.9% vs. 21.0%, p =0.41), respectively. The aPL titers were also similar regardless of thrombosis. The levels of β2GPI in COVID-19 patients were lower than those in the healthy donors., Conclusion: Although aPLs were frequently detected in Japanese COVID-19 patients, their prevalence and titer were irrelevant to thrombotic complications. While COVID-19 patients have lower levels of serum β2GPI than healthy blood donors, β2GPI levels were indifferent regardless of thrombosis. Although most of the titers were below cut-offs, positive correlations were observed among aPLs, suggesting that the immune reactions against aPL antigens were induced by COVID-19. We should focus on the long-term thromboembolic risk and the development of APS in the aPL-positive patients with high titer or multiple aPLs., Competing Interests: SY received research funding from Abbvie, Asahi Kasei, Pharma, Chugai Pharmaceutical, CSL Behring, Eisai, ImmunoForge, Mitsubishi Tanabe, Pharma, and Ono Pharmaceutical, speaking fees from Abbvie, Asahi Kasei Pharma, Chugai Pharmaceutical, Eisai, Eli Lilly, GlaxoSmithKline, Mitsubishi Tanabe Pharma, Ono pharmaceutical, and Pfizer. YM received a research grant and an honorarium from Chugai Pharmaceutical Co., Ltd. TH received a research grant from Sony corporation. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Oba, Hosoya, Kaneshige, Kawata, Yamaguchi, Mitsumura, Shimada, Shibata, Tateishi, Koike, Tohda, Hirakawa, Yoko, Otomo, Nojima, Miyazaki and Yasuda.)

Methotrexate (MTX)-associated lymphoproliferative disorder (MTX-LPD) is a troublesome problem in patients receiving MTX for rheumatoid arthritis (RA). However, its incidence, prognosis, and risk factors remain unclear. In this retrospective study, we evaluated the actual incidence, prognostic impact, and risk factors of MTX-LPD. Of the 986 patients with RA treated with MTX, 90 patients experienced 95 new malignancies (NMs), with LPD as the most frequent in 26 patients. The cumulative LPD incidences were 1.3% and 4.7% at 5 and 10 years after MTX initiation, respectively. Among the 24 patients who discontinued MTX after developing LPD, 15 showed sustained regression, without difference in overall survival between patients with LPD and without NM. Inflammatory markers and absolute lymphocyte counts were not useful for early LPD development detection, but most of the patients with LPD had persistently elevated erythrocyte sedimentation ratios. Regarding concomitant drugs, tacrolimus increased the risk only if patients were not receiving biological disease-modifying antirheumatic drugs (bDMARDs). bDMARDs did not increase the risk for any of the drugs or the number of classes used. The number of LPD cases was lower in patients with IL-6A even after a long period after MTX, although with no statistically significant difference. Thus, approximately 1 in 20 patients with RA developed MTX-LPD over the 10 years of MTX treatment, but it did not affect the survival of patients with RA. Tacrolimus increased the risk of developing LPD for certain patients and should be used with caution., (© 2023 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Background: Sublineage BA.5 of the SARS-CoV-2 Omicron variant rapidly spread and replaced BA.2 in July 2022 in Tokyo. A high viral load can be a possible cause of high transmissibility., Methods and Results: The copy numbers of SARS-CoV-2 in nasopharyngeal swab samples obtained from all patients visiting the hospital where this research was conducted were measured using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Viral genotypes were determined using PCR-based melting curve analysis. Next, whole-genome sequencing was performed using approximately one fifth of the samples to verify the viral genotypes determined using PCR. Then, the copy numbers of the BA.1, BA.2, and BA.5 cases were compared. Contrary to expectations, the copy numbers of the BA.5 cases (median 4.7 × 10 4 copies/μL, n = 291) were significantly (p = .001) lower than those of BA.2 cases (median 1.1 × 10 5 copies/μL, n = 184). There was no significant difference (p = .44) between the BA.5 and BA.1 cases (median, 3.3 × 10 4 copies/μL; n = 215)., Conclusion: The results presented here suggest that the increased infectivity of BA.5 is not caused by higher viral loads, but presumably by other factors such as increased affinity to human cell receptors or immune escape due to its L452R mutation., (© 2023 The Authors. Immunity, Inflammation and Disease published by John Wiley & Sons Ltd.)

Factors associated with mortality are important in the treatment of coronavirus disease 2019 (COVID-19). Polymerase chain reaction (PCR) is the gold standard for diagnosing COVID-19, which reflects the viral load in the upper respiratory tract. In total, 523 patients were enrolled in this study; of them, 441 and 75 patients underwent PCR testing of nasopharyngeal swabs and sputum samples, respectively, within 20 days from onset of COVID-19. We investigated the association between RNA copy number and the COVID-19 severity and mortality rate and its effect on the predictive performance for severity and mortality. RNA copy numbers in nasopharyngeal swabs were higher in the non-survivor group than in the survivor group. Multivariate logistic regression analysis identified that the high RNA copy number (≥9 log 10 /swab) in nasopharyngeal swabs was a factor associated with mortality (odds ratio, 4.50; 95% confidence interval, 1.510-13.100; P = 0.008). Furthermore, adding RNA copy number (≥9 log 10 /swab) in severe cases, adjusted by duration from onset to PCR, improved mortality predictive performance based on known factors. The RNA copy number is a factor associated with the mortality of patients with COVID-19 and can improve the predictive performance of mortality in severe cases.

Carbapenem-resistant bacteria represent an emerging threat to global health; nursing homes may be reservoirs for these isolates, which cause life-threatening infections. Here, we present the complete genome sequence of an Enterobacter roggenkampii strain with reduced carbapenem susceptibility that was isolated from a sink in a home-visit nursing agency.

Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge 1-5 . Here we conducted a genome-wide association study (GWAS) involving 2,393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3,289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target., (© 2022. The Author(s).)

Coronavirus disease 2019 (COVID-19) is a recently-emerged infectious disease that has caused millions of deaths, where comprehensive understanding of disease mechanisms is still unestablished. In particular, studies of gene expression dynamics and regulation landscape in COVID-19 infected individuals are limited. Here, we report on a thorough analysis of whole blood RNA-seq data from 465 genotyped samples from the Japan COVID-19 Task Force, including 359 severe and 106 non-severe COVID-19 cases. We discover 1169 putative causal expression quantitative trait loci (eQTLs) including 34 possible colocalizations with biobank fine-mapping results of hematopoietic traits in a Japanese population, 1549 putative causal splice QTLs (sQTLs; e.g. two independent sQTLs at TOR1AIP1), as well as biologically interpretable trans-eQTL examples (e.g., REST and STING1), all fine-mapped at single variant resolution. We perform differential gene expression analysis to elucidate 198 genes with increased expression in severe COVID-19 cases and enriched for innate immune-related functions. Finally, we evaluate the limited but non-zero effect of COVID-19 phenotype on eQTL discovery, and highlight the presence of COVID-19 severity-interaction eQTLs (ieQTLs; e.g., CLEC4C and MYBL2). Our study provides a comprehensive catalog of whole blood regulatory variants in Japanese, as well as a reference for transcriptional landscapes in response to COVID-19 infection., (© 2022. The Author(s).)

Neisseria meningitidis causes a life-threatening invasive meningococcal disease (IMD). Isolates resistant to antibiotics, such as penicillin, ceftriaxone, and ciprofloxacin that are recommended for the treatment of IMD patients and their close contacts have been serious public health concerns globally. However, susceptibility profiles to critically important antibiotics and the genetic characteristics of isolates possessing antibiotic resistance are extremely limited as IMD incidence is low in Japan. We assessed the susceptibility profiles of 87 randomly selected, sterile site-derived N. meningitidis strains isolated from hospitals nationwide, recovered between April 1998 and March 2018 in Japan, to seven antibiotics. As a result, we demonstrated, for the first time, that the isolates remained highly susceptible to ceftriaxone, meropenem, azithromycin, ciprofloxacin, chloramphenicol, and rifampin, but not to penicillin. We then characterized the genetic relatedness of six penicillin- and/or ciprofloxacin-resistant isolates obtained in this study with global 112 genomes using core-genome phylogenetic analysis. These results provide the first evidence that invasive lineages such as a penicillin-resistant serogroup W, sequence type (ST)-11 clonal complex (CC), and a ciprofloxacin-resistant serogroup B/C, ST-4821 CC that is considered as a global threat, have been sporadically identified in Japan. Our findings highlight the need to monitor antibiotic resistance in clinical isolates of N. meningitidis, thereby preventing the spread of antibiotic-resistant invasive lineages and maintaining effective treatment for IMD patients and their close contacts. IMPORTANCE Although antibiotics such as penicillin and ceftriaxone can treat invasive meningococcal disease (IMD), the emergence and spread of antibiotic-resistant Neisseria meningitidis have become a global concern. To provide effective treatment, including chemoprophylaxis to IMD patients and their close contacts, we highlighted the importance of recognizing the antibiotic resistance and genetic features of N. meningitidis isolates.

Background: Thrombosis is a characteristic complication in coronavirus disease 2019 (COVID-19). Since coagulopathy has been observed over the entire clinical course, thrombosis might be a clue to understanding the specific pathology in COVID-19. Currently, there is limited epidemiological data of COVID-19-associated thrombosis in the Japanese population and none regarding variant strains of SARS-CoV-2. Here, we elucidate the risk factors and the pattern of thrombosis in COVID-19 patients. Methods: The patients consecutively admitted to Tokyo Medical and Dental University Hospital with COVID-19 were retrospectively analyzed. SARS-CoV-2 variants of concern/interest (VOC/VOI) carrying the spike protein mutants E484K, N501Y, or L452R were identified by PCR-based analysis. All thrombotic events were diagnosed by clinical symptoms, ultrasonography, and/or radiological tests. Results: Among the 516 patients, 32 patients experienced 42 thromboembolic events. Advanced age, severe respiratory conditions, and several abnormal laboratory markers were associated with the development of thrombosis. While thrombotic events occurred in 13% of the patients with a severe respiratory condition, those events still occurred in 2.5% of the patients who did not require oxygen therapy. Elevated D-dimer and ferritin levels on admission were independent risk factors of thrombosis (adjusted odds ratio 9.39 and 3.11, 95% confidence interval 2.08-42.3, and 1.06-9.17, respectively). Of the thrombotic events, 22 were venous, whereas 20 were arterial. While patients with thrombosis received anticoagulation and antiinflammatory therapies with a higher proportion, the mortality rate, organ dysfunctions, and bleeding complications in these patients were higher than those without thrombosis. The incidence of thrombosis in COVID-19 became less frequent over time, such as during the replacement of the earlier strains of SARS-CoV-2 by VOC/VOI and during increased use of anticoagulatory therapeutics. Conclusion: This study elucidated that elevated D-dimer and ferritin levels are useful biomarkers of thrombosis in COVID-19 patients. The comparable incidence of arterial thrombosis with venous thrombosis and the development of thrombosis in less severe patients required further considerations for the management of Japanese patients with COVID-19. Further studies would be required to identify high-risk populations and establish appropriate interventions for thrombotic complications in COVID-19., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Oba, Hosoya, Amamiya, Mitsumura, Kawata, Sasaki, Kamiya, Yamamoto, Ando, Shimada, Shirai, Okamoto, Tateishi, Endo, Aiboshi, Nosaka, Yamanouchi, Ugawa, Nagaoka, Oi, Tao, Maejima, Tanaka, Tanimoto, Takeuchi, Tohda, Hirakawa, Sasano, Arai, Otomo, Miyazaki and Yasuda.)

Background/aim: Homeobox A9 (HOXA9), a transcription factor regulating haematopoiesis and leukaemia cell proliferation, is suggested as a driver of acute myeloid leukaemia (AML). The aim of this study was to examine the effects of a synthetic HOXA9 inhibitor DB818 on AML cells in vitro., Materials and Methods: AML cell lines OCI/AML3, MV4-11, and THP-1 with gene mutations up-regulating HOXA9 expression were treated with DB818 and analysed for cell proliferation and gene expression. The effects of HOXA9 knockdown were also evaluated., Results: In the three AML cell lines, DB818 suppressed growth, induced apoptosis, and down-regulated the expression of HOXA9 transcriptional target genes: MYB proto-oncogene, transcription factor (MYB), MYC proto-oncogene, bHLH transcription factor (MYC), and BCL2 apoptosis regulator (BCL2), while up-regulating that of Fos proto-oncogene, AP-1 transcription factor subunit (FOS). HOXA9 knockdown showed similar effects, except for MYC expression, which differed between DB818-treated and HOXA9-deficient OCI/AML3 cells, suggesting an off-target effect of DB818., Conclusion: DB818 has potential as a novel molecular targeted drug for treating AML associated with HOXA9 overexpression., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

High-density lipoprotein- (HDL-) cholesterol measurements are generally used in the diagnosis of cardiovascular diseases. However, HDL is a complicated heterogeneous lipoprotein, and furthermore, it can be converted into dysfunctional forms during pathological conditions including inflammation. Therefore, qualitative analysis of pathophysiologically diversified HDL forms is important. A recent study demonstrated that serum amyloid A (SAA) can remodel HDL and induce atherosclerosis not only over long periods of time, such as during chronic inflammation, but also over shorter periods. However, few studies have investigated rapid HDL remodeling. In this study, we analyzed HDL samples from patients undergoing orthopedic surgery inducing acute inflammation. We enrolled 13 otherwise healthy patients who underwent orthopedic surgery. Plasma samples were obtained on preoperative day and postoperative days (POD) 1-7. SAA, apolipoprotein A-I (apoA-I), and apolipoprotein A-II (apoA-II) levels in the isolated HDL were determined. HDL particle size, surface charge, and SAA and apoA-I distributions were also analyzed. In every patient, plasma SAA levels peaked on POD3. Consistently, the HDL apoA-I : apoA-II ratio markedly decreased at this timepoint. Native-polyacrylamide gel electrophoresis and high-performance liquid chromatography revealed the loss of small HDL particles during acute inflammation. Furthermore, HDL had a decreased negative surface charge on POD3 compared to the other timepoints. All changes observed were SAA-dependent. SAA-dependent rapid changes in HDL size and surface charge were observed after orthopedic surgery. These changes might affect the atheroprotective functions of HDL, and its analysis can be available for the qualitative HDL assessment., Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (Copyright © 2021 Shitsuko Shimano et al.)

Background/aim: The deacetylase sirtuin1 (SIRT1) inhibits tumor suppressor p53 and may promote tumorigenesis; however, SIRT1 effects on leukemia cells are controversial. The aim of this study was to clarify the activity of SIRT1 in leukemia cells., Materials and Methods: The effects of SIRT1 inhibition or activation and SIRT1 knockdown or overexpression were examined in two T cell acute lymphoblastic leukemia (T-ALL) cell lines carrying NOTCH1 mutations and three acute myeloid leukemia (AML) cell lines., Results: The growth of T-ALL cells was promoted by SIRT1 inhibition and SIRT1 knockdown but was reduced by SIRT1 activation and overexpression; however, no effects were observed in AML cells. SIRT1 activation decreased NOTCH, NF-κB, and mTOR signaling and inhibited p53, suggesting that the possible mechanisms of T-ALL growth suppression by SIRT1 are independent of p53., Conclusion: SIRT1 activators acting through the down-regulation of NOTCH, NF-κB, and mTOR pathways can be novel targeted drugs for NOTCH1-mutated T-ALLs., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Purpose: Bile acids play an important role in Clostridioides difficile life cycle. Deoxycholate (DCA), one of the most abundant secondary bile acids, is known to inhibit vegetative growth and toxin production. However, limited data are available on the role of DCA on C. difficile sporulation. Here, we investigated the phenotypic and genotypic impact of DCA on the growth, toxin production, and sporulation of C. difficile ., Methodology: Four genetically divergent C. difficile strains were cultured in nutrient-rich broth with and without DCA at various concentrations, and growth activity was evaluated for each strain. Cytotoxicity assays using culture supernatants from cells grown in nutrient-rich broth with and without 0.01% DCA were conducted. Sporulation efficiency was determined using sporulation media with and without 0.01% DCA. Transcript levels of tcdB and spo0A were analyzed using quantitative reverse-transcription polymerase chain reaction., Results: We found that DCA led to growth reduction in a dose-depended manner and regulated toxin production by repressing tcdB expression during vegetative growth. To our knowledge, we have also provided the first evidence that DCA reduces C. difficile sporulation efficiency through the downregulation of spo0A expression during the sporulation stage., Conclusions: DCA modulates C. difficile sporulation, vegetative growth, and toxin production., (© 2020 The Author(s).)

Clostridioides difficile hypervirulent clade 2 lineages are the major lineages responsible for the outbreaks in North America and Europe. However, the genome sequences of Japanese isolates are scarcely available. Herein, we report the draft genome sequence of C. difficile strain TMD0138 sequence type 97 (ST97), belonging to hypervirulent clade 2, isolated in Japan., (Copyright © 2020 Usui et al.)

Background: Clostridioides difficile ribotype (RT) 019/sequence type (ST) 67 strains belong to a hypervirulent lineage closely related to RT027/ST1; however, limited data are available for hypervirulent clade 2 lineages in Japan. Herein, we report the draft genome of a C. difficile strain B18-123 belonging to clade 2, RT019/ST67 for the first time in Japan., Results: The pathogenicity locus carried by B18-123 (19.6 kb) showed higher homology (97.29% nucleotide identity) with strain R20291 (RT027/ST1) than the reference strain 630 (RT012/ST54), and B18-123 harbored 8-nucleotide substitutions in tcdC . However, it did not contain an 18-base pair (bp) deletion or a single-bp deletion at position 117 in tcdC , which was identified in the previous strain R20291. A cytotoxicity assay revealed similar cytotoxicity levels between strains B18-123 and ATCC BAA-1870 (RT027/ST1). The B18-123 strain was found to be susceptible to metronidazole and vancomycin., Conclusion: Our findings contribute to the further understanding of the characteristics of hypervirulent clade 2 including RT019/ST67 lineages., Competing Interests: Competing interestsThe authors declare that they have no competing interests., (© The Author(s) 2019.)

Aim: To examine the influence of hypoxia on the in vitro growth of leukaemia cells and the activity of signalling proteins to better understand the pathophysiology of leukaemia cells in human bone marrow., Materials and Methods: Six human leukaemia cell lines were cultured under normoxic or hypoxic conditions. Cell growth, recovery of clonogenic cells, and the expression and activation of various signalling proteins were examined., Results: Hypoxia suppressed cell growth and the recovery of clonogenic cells. Moreover, hypoxia up-regulated hypoxia-inducible factor (HIF) 1α and HIF2α expression while suppressing the expression and activation of NOTCH1, mechanistic target of rapamycin kinase (mTOR) activation, and nuclear factor-kappa B (NF-κB) phosphorylation., Conclusion: We found that hypoxia up-regulated HIF expression while it suppressed the self-renewal capacity of leukaemia cells, NOTCH activity, and expression of its down-stream signalling molecules, which differs from previous reports mentioning that HIF activates NOTCH signalling. Our findings serve to further elucidate the in vivo pathophysiology of leukaemia cells., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Cholesterol efflux capacity (CEC) in atherosclerotic lesions is the main anti-atherosclerotic function of high-density lipoprotein (HDL). In recent studies, apolipoprotein (apo) B-depleted serum (BDS) obtained with the polyethylene glycol (PEG) precipitation method is used as a cholesterol acceptor (CA) substitution for HDL isolated by ultracentrifugation. However, the suitability of BDS as a CA is controversial. In the present study, CEC obtained from BDS (BDS-CEC) was evaluated based on a parameter, defined as whole-CEC, which was calculated by multiplying CEC obtained using fixed amounts of HDL by cholesterol concentration to HDL-cholesterol (HDL-C) levels in the serum. Significant correlation (r = 0.633) was observed between both CECs. To eliminate systematic errors from possible contamination with serum proteins and low-density lipoprotein (LDL) or very-LDL (VLDL) in BDS-CEC, the deviation of each CEC-BDS from the regression equation was compared with serum protein, LDL, and triglyceride (TG) levels. No correlation was observed between the deviation and the levels of each of these serum components, indicating that the deviations do not derive from systematic error. Further, to evaluate the effects of serum protein on the results, we measured BDS-CEC of reconstituted serum samples prepared using combinations of five levels of serum proteins with five levels of HDL-C. No significant change in BDS-CEC was observed in any combination. These results indicate that BDS-CEC reflects not only the function of HDL but also its concentration in serum., (© 2019 The Author(s).)

Background: This study examined whether and how chloroquine inhibits blast progenitor self-renewal (SR) synergistically with phytochemicals or nonsteroidal anti-inflammatory drugs in seven hematological malignant cell lines., Materials and Methods: Vitamin C, resveratrol, cyclo-oxygenase inhibitor NS-398 and indomethacin heptyl ester (Ind) were added to cell culture with or without 3 μM chloroquine., Results: Chloroquine synergistically inhibited blast colony formation in methylcellulose with vitamin C, resveratrol, NS-398 and Ind in one, two, none and one cell lines, respectively, in a total of four out of 28 conditions. Chloroquine synergistically inhibited blast progenitor SR in suspension with vitamin C, resveratrol, NS-398 and Ind in four, six, one and five cell lines, respectively, in a total of 16 out of 28 conditions. In contrast, chloroquine abolished SR inhibition by another agent in four out of 28 conditions., Conclusion: Chloroquine exerted a marked synergistic inhibition of blast progenitor SR, but not blast colony formation., (Copyright© 2019, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background: Hedgehog (HH), WNT, NOTCH, and mechanistic target of rapamycin (mTOR) signalling pathways are known to regulate the progression of cancer; however, their interaction in leukaemia cells is not fully clarified., Materials and Methods: Myeloid and T-lymphoblastic leukaemia cell lines (NB4, THP-1, Jurkat, and DND-41) were transfected with small interfering RNAs targeting the glioma-associated oncogene homolog 1 (GLI1) and catenin beta-1 (CTNNB1) genes involved in the regulation of HH and WNT pathways, respectively, and we examined cell proliferation and gene expression., Results: The knockdown of GLI1 and CTNNB1 did not significantly affect proliferation of any cell line; however, it up-regulated the expression of NOTCH1, cleaved NOTCH1 fragment, and phosphorylated mTOR in NB4 cells, but not in the other cell lines., Conclusion: Our data suggest that HH and WNT act upstream of NOTCH and mTOR pathways and negatively regulate them in myeloid NB4 cells. Further studies are required to determine the biological significance of this signalling crosstalk in leukaemia., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

An easy-to-use assessment for activated factor X (FXa) is lacking despite its pivotal role in the coagulation. Dielectric blood coagulometry (DBCM) was recently invented as a novel assessment tool for determining the whole blood coagulability by measuring the temporal change in the permittivity of blood. We previously reported that it could evaluate the global blood coagulability. This study aimed to apply the DBCM for assessing FXa activity and its inhibition by anticoagulants. We performed the DBCM analysis along with measurement of the FXa activity by a fluorometric assay in samples from healthy subjects, and identified a new index named maximum acceleration time (MAT) that had a correlation to the FXa activity. Next the DBCM analysis was performed using blood samples mixed with anticoagulants (unfractionated heparin, dalteparin, and edoxaban). Blood samples with three anticoagulants had different profiles of the temporal change in the permittivity, reflecting their different selectivity for FXa. We compared the MAT with the anti-FXa activity assay, and found that the prolongation of MAT was similarly correlated with the anti-FXa activity regardless of the type of anticoagulants. In conclusion, the DBCM has the possibility for evaluating the innate FXa activity and effect of anticoagulants focusing on their FXa inhibition.

NOTCH signaling diversely regulates the growth of acute myeloid leukemia (AML) cells. It is known that glycosylation of NOTCH receptors modulates NOTCH activation. However, little is known about glycosylation of NOTCH in AML cells. We examined the effects of ligand-induced NOTCH activation on the expression of NOTCHmodifying glycosyltransferases in two AML cell lines, THP-1 and TMD7. The cells were stimulated with recombinant NOTCH ligands JAGGED1 and DELTA1, and subjected to immunoblot analysis to evaluate the expression levels of glycosyltransferases. Ligand stimulation promoted the expression of POFUT1, LFNG, MFNG, RFNG, GXYLT1, GXYLT2, and XXYLT1 in THP-1 cells, and that of RFNG and GXYLT1 in TMD7 cells. We found that NOTCH activation promoted the expression of several glycosyltransferases in AML cells. This suggests that NOTCH activation modulates its sensitivity to NOTCH ligands by increased glycosylation of NOTCH receptors in AML cells. Further investigation is needed to elucidate its biological significance., Competing Interests: Conflict of interests: the authors declare no conflict of interests.

Objective: Forkhead box P3 (FOXP3) is a master transcriptional factor of regulatory T-cells (Tregs). Recent studies have shown that FOXP3 is associated with growth inhibition of cancer cells. However, the role of FOXP3 in acute T-lymphoblastic leukemia (T-ALL) cells is not known. It was also reported that NOTCH signaling promoted the expression of FOXP3 in Tregs. However, the effect of FOXP3 on NOTCH expression in T-ALL cells is little known. Therefore, we examined the effect of FOXP3 knockdown on the proliferation of T-ALL cells and NOTCH1 signaling., Results: Two T-ALL cell lines Jurkat and KOPT-K1, harboring activating NOTCH1 mutations, were transfected with small interfering RNA against FOXP3. Cell growth was assessed with a colorimetric assay and morphology was observed under a microscope. FOXP3 knockdown significantly reduced cell growth and induced morphological changes suggesting apoptosis. Quantitative polymerase chain reaction revealed that FOXP3 knockdown caused the downregulation of mRNA expression of NOTCH1 and HES1. These findings suggest that FOXP3 supports the growth of T-ALL cells although this can not be generalized because we examined only two cell lines. The observed growth suppression can be partly due to the downregulation of NOTCH1 signaling. FOXP3 may be a potential therapeutic target in T-ALL.

Estimation of the function as well as the amount of high-density lipoprotein (HDL) is required to predict the risk of cardiovascular disease development. Cholesterol efflux capacity (CEC) is the key metric for determining the antiatherosclerotic function of HDL. However, the assay methods currently used to calculate CEC are not ideal for clinical use as they require the culture of cells. In the present study, we developed a novel CEC assay using immobilized liposome-bound gel beads (ILGs), containing fluorescently labeled cholesterol, as a substitute for cultured cells. When apolipoprotein B-100 depleted serum, obtained by polyethylene glycol precipitation, was used as the cholesterol acceptors, the basic properties of this method, such as the available range of HDL-cholesterol, efflux temperature and time, and normalization parameters, indicate that this method is sufficient to estimate CEC. Furthermore, the CEC values obtained with this ILG method were also correlated with those obtained with a conventional method using THP-1 macrophages derived foam cells and 3 H-cholesterol as a tracer ( r = 0.932). Overall, this novel cholesterol efflux assay method is a realistic and effective alternative to current methods in the field while also being easier to use in clinical laboratories as neither cell culture, radioisotope nor ultracentrifugation is required., (© 2018 The Author(s).)

Background: MER proto-oncogene tyrosine kinase (MERTK) is a receptor tyrosine kinase that affects cancer cell proliferation. This study evaluated the effects of the synthetic MERTK inhibitors UNC569 and UNC1062 on in vitro growth of acute myeloid leukaemia (AML) cells., Materials and Methods: Four AML cell lines expressing MERTK were treated with UNC569 and UNC1062 and analyzed for cell proliferation, immunoblotting, and gene expression. The effects of MERTK knockdown were also evaluated., Results: Treatment with the inhibitors suppressed cell growth and induced apoptosis in all cell lines. OCI/AML5 and TMD7 cells, in which MERTK was constitutively phosphorylated by autocrine mechanisms, were highly susceptible to these inhibitors. The treatment reduced the phosphorylation of MERTK and its down-stream signalling molecules, v-akt murine thymoma viral oncogene homolog 1 (AKT) and extracellular signal-regulated kinase (ERK). Similar effects were observed after MERTK knockdown. The inhibitors and the knockdown caused similar changes in mRNA expression., Conclusion: These MERTK inhibitors are potential molecular-targeted drugs for treating AML expressing constitutively phosphorylated MERTK., (Copyright© 2018, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Myeloid differentiation primary response gene 88 (MYD88), which activates the nuclear factor kappa B (NF-κB) pathway, is important for the growth of lymphoma and leukaemia cells. In this study, we investigated the effects of ST2825, a synthetic peptidomimetic compound which inhibits MYD88 homodimerization, on their growth., Materials and Methods: Seven lymphoma and leukaemia cell lines including TMD8, a B-cell lymphoma line with MYD88-activating mutation, were treated with ST2825 and analysed for cell proliferation and expression of NF-κB signalling-related molecules., Results: ST2825 suppressed the growth of all cell lines by inducing apoptosis and down-regulating phosphorylation of NF-κB pathway components inhibitor of nuclear factor kappa B kinase (IκB) and reticuloendotheliosis oncogene A (RelA), as well as of MYD88 activator Bruton tyrosine kinase (BTK), suggesting that MYD88 may affect BTK activity. ST2825 effects were specific as MYD88-targeting siRNA also suppressed phosphorylation of NF-κB signalling proteins and BTK in TMD8 cells., Conclusion: ST2825 may be a novel drug targeting not only B-lymphoid malignancies with MYD88 mutations, but also lymphoma and leukaemia with wild-type MYD88., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: B cell-specific Moloney murine leukemia virus integration site 1 (BMI1) is up-regulated in several cancers; therefore, we investigated the effects of BMI1 inhibitors on leukemia cells., Materials and Methods: Four acute myeloid leukemia and two T-lymphoblastic leukemia cell lines were treated with BMI1 inhibitors artemisinin, PRT4165, and PTC-209 and analyzed for cell proliferation and gene expression by microarray and immunoblotting., Results: PTC-209 and PRT4165 suppressed the growth of all cell lines through apoptosis.Artemisinin acted only on Jurkat cells. BMI1 inhibitors and BMI1-specific siRNA down-regulated the expression of NOTCH signaling proteins NOTCH1, HES1, and MYC. All but one cell lines did not have the cyclin-dependent kinase inhibitor 2A (CDKN2A) gene targeted by BMI1, thus the inhibitors acted through CDKN2A-independent pathways., Conclusion: BMI1 inhibition suppressed proliferation of leukemia cells through NOTCH signaling which functions downstream of BMI1, suggesting that BMI1 inhibitors can be candidate targeted drugs against leukemia., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Background/aim: Hedgehog (HH) and NOTCH pathways are involved in the regulation of cancer stem cells and haematopoietic malignancies. However, the effects of HH stimulation on cell growth and NOTCH signalling in acute T-lymphoblastic leukaemia (T-ALL) cells have not been elucidated., Materials and Methods: Two T-ALL cell lines, Jurkat and KOPT-K1 harbouring activating NOTCH1 mutations, were cultured with recombinant Sonic (S) HH and analysed for proliferation, colony formation, and expression of NOTCH-regulated genes and proteins., Results: SHH stimulation did not affect cell growth but suppressed colony formation, increased the levels of cleaved NOTCH1 fragment characteristic for NOTCH1 activation, and upregulated mRNA expression of HES1, while decreasing that of MYC in Jurkat cells. However, no such effects were observed in KOPT-K1 cells., Conclusion: Our results indicate that SHH stimulation activates NOTCH signalling in Jurkat cells, thus disclosing a novel relationship between HH and NOTCH pathways., (Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Nucleophosmin (NPM1) mutations, generally consisting of a four base-pair insertion, are present in ~60% of all cytogenetically normal acute myeloid leukemia (AML) cases. The mutation is clinically significant as an important prognostic factor. Direct sequencing is the current standard method of mutation detection, however, it is quite costly and time consuming. The present study aimed to establish a highly sensitive quenching probe (QP) method to detect NPM1 mutations efficiently. Melting curve analysis was performed using a QP, following polymerase chain reaction for amplification of the involved region of the gene. The curve derived from the fluorescent intensity with respect to the temperature of OCI/AML3, a heterozygous NPM1 mutant AML cell line, was W-shaped with melting peaks at 61°C and 68°C. That of M-07e, the homozygous wild type cell line, was V-shaped with a melting peak at 68°C. Thus, the curve derived from the mutant allele was easily discriminated from that of the wild-type allele. The mutant allele was detected in concentrations as low as 3% as determined by a subsequent sensitivity study. With a short testing time and a high sensitivity, this assay was applicable for NPM1-mutated AML patient samples and is appropriate for screening NPM1 mutations. It does require further examination as to whether it would be useful as a detection method for other mutant alleles since NPM1 mutations may consist of 61 known types of mutant sequences. To the best of our knowledge, this is the first report describing the QP method for the detection of NPM1 mutations.

A 68-year-old man was admitted because of weakness of the left leg, dysesthesiae of the extremities and bilateral lower extremity purpura. A neurological examination showed mononeuritis multiplex with laboratory evidence of hypocomplementemia, cryoglobulinemia and leukocytoclastic vasculitis in the biopsy of a skin specimen. The patient also exhibited bilateral submandibular gland swelling, elevated serum IgG4 levels and infiltration of a large number of IgG4-positive plasma cells in the submandibular glands. These findings were consistent with both cryoglobulinemic vasculitis and IgG4-related disease. The administration of oral prednisolone (1 mg/kg/day) resolved the neurological manifestations and the swelling of the submandibular glands and cryoglobulinemia.

NOTCH activation plays oncogenic roles in acute T-lymphoblastic leukaemia (T-ALL). However, whether NOTCH is oncogenic or tumor-suppressive in acute myeloid leukaemia (AML) is still controversial. Herein, the roles of NOTCH in AML are reviewed. AML cells express NOTCH and NOTCH ligands; however, cell-autonomous activation is not observed. Activating NOTCH1 mutations are rare in AML, unlike in T-ALL. NOTCH ligand stimulation generally suppresses the in vitro growth of AML cells but promotes transient growth of some samples. Conversely, knockdown of NOTCH1 and NOTCH2 does not affect the growth of AML cells, whereas it suppresses the growth of T-ALL cells. These findings suggest that NOTCH is dispensable or suppressive for AML cell growth. However, the effects of NOTCH differ depending on cell conditions, and various stemness-related signals modify these effects; hence, forced NOTCH activation in vitro may not exhibit effects in bone marrow. Thus, further understanding is required for the development of AML therapies targeting NOTCH signalling., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

Background: Signaling induced by binding of erythropoietin-producing hepatoma-amplified sequence (EPH) receptors to their cell-surface ephrin ligands is implicated in hematopoiesis and growth of various cancer cells. However, the roles of EPH-ephrin signaling in leukemia have not been elucidated. We investigated the effects of EPHB4 and ephrin B2 on the growth of leukemia cells., Materials and Methods: Seven human leukemia cell lines were used to examine the effects of recombinant ephrin B2 and EPHB4 on cell proliferation by colorimetric WST-1 assay and colony assays; on protein tyrosine phosphorylation; and on mRNA expression by reverse transcription-polymerase chain reaction and microarray analysis., Results: In an erythroid leukemia-derived cell line AA, exogenous ephrin B2 induced proliferation and colony formation; in addition, it up-regulated protein tyrosine phosphorylation and the expression of growth-related genes such as FBJ murine osteosarcoma viral oncogene homolog B and v-src avian sarcoma viral oncogene homolog., Conclusion: Growth-promoting effects of ephrin B2 were observed in an erythroid leukemia cell line, suggesting that the EPH-ephrin signaling may be involved in the pathology of leukemia., (Copyright© 2014 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved.)

We report a case of catheter-related bloodstream infection by Tsukamurella inchonensis, identified using 16S rRNA gene sequencing, in a patient with myelofibrosis who underwent a bone marrow transplant. Tsukamurella species infections are rare. To our knowledge, this is the first case of T. inchonensis bloodstream infection in an immunocompromised patient., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

We investigated a novel Japanese isolate of sequence type 11 (ST11), the Klebsiella pneumoniae carbapenemase-2 (KPC-2)-producing K. pneumoniae strain Kp3018, which was previously obtained from a patient treated at a Brazilian hospital. This strain was resistant to various antibiotic classes, including carbapenems, and harbored the gene blaKPC-2, which was present on the transferable plasmid of ca. 190 kb, in addition to the blaCTX-M-15 gene. Furthermore, the ca. 2.3-kb sequences (ISKpn8-blaKPC-2-ISKpn6-like), encompassing blaKPC-2, were found to be similar to those of K. pneumoniae strains from China.

The gain of function mutation JAK2-V617F is very frequently found in myeloproliferative neoplasms (MPNs) and is strongly implicated in pathogenesis of these and other hematological malignancies. Here we report establishment of a new leukemia cell line, PVTL-1, homozygous for JAK2-V617F from a 73-year-old female patient with acute myeloid leukemia (AML) transformed from MPN. PVTL-1 is positive for CD7, CD13, CD33, CD34, CD117, HLA-DR, and MPO, and has complex karyotypic abnormalities, 44,XX,-5q,-7,-8,add(11)(p11.2),add(11)(q23),-16,+21,-22,+mar1. Sequence analysis of JAK2 revealed only the mutated allele coding for Jak2-V617F. Proliferation of PVTL-1 was inhibited and apoptosis was induced by the pan-Jak inhibitor Jak inhibitor-1 (JakI-1) or dasatinib, which inhibits the Src family kinases as well as BCR/ABL. Consistently, the Src family kinase Lyn was constitutively activated with phosphorylation of Y396 in the activation loop, which was inhibited by dasatinib but not by JakI-1. Further analyses with JakI-1 and dasatinib indicated that Jak2-V617F phosphorylated STAT5 and SHP2 while Lyn phosphorylated SHP1, SHP2, Gab-2, c-Cbl, and CrkL to induce the SHP2/Gab2 and c-Cbl/CrkL complex formation. In addition, JakI-1 and dasatinib inactivated the mTOR/p70S6K/4EBP1 pathway and reduced the inhibitory phosphorylation of GSK3 in PVTL-1 cells, which correlated with their effects on proliferation and survival of these cells. Furthermore, inhibition of GSK3 by its inhibitor SB216763 mitigated apoptosis induced by dasatinib but not by JakI-1. Together, these data suggest that apoptosis may be suppressed in PVTL-1 cells through inactivation of GSK3 by Lyn as well as Jak2-V617F and additionally through activation of STAT5 by Jak2-V617F. It is also speculated that activation of the mTOR/p70S6K/4EBP1 pathway may mediate proliferation signaling from Jak2-V617F and Lyn. PVTL-1 cells may provide a valuable model system to elucidate the molecular mechanisms involved in evolution of Jak2-V617F-expressing MPN to AML and to develop novel therapies against this intractable condition.

Aim: The effects of small interfering RNA (siRNA)-mediated knockdown of NOTCH1 and NOTCH2 on cell proliferation and downstream signaling pathways in leukemia cells were examined., Materials and Methods: Two T-lymphoblastic leukemia (T-ALL) cell lines and two acute myeloblastic leukemia (AML) cell lines were transfected with siRNAs targeting NOTCH1 and NOTCH2. The effects of knockdown on cell proliferation and protein expression were examined by colorimetric WST-8 assay and immunoblotting, respectively., Results: In T-ALL cell lines, NOTCH1 knockdown as well as NOTCH2 knockdown suppressed cell proliferation and induced apoptosis. v-Myc avian myelocytomatosis viral oncogene homolog (MYC) protein expression was down-regulated in NOTCH1-knockdown cells but not affected in NOTCH2-knockdown cells. In AML cell lines, cell proliferation was not significantly affected by NOTCH siRNAs. NOTCH2 knockdown increased the level of cleaved NOTCH1 fragment without increasing NOTCH1 expression. NOTCH knockdown reduced the level of mechanistic target of rapamycin (mTOR) protein in the monoblastic leukemia cell line THP-1. Contrastingly, NOTCH activation by NOTCH ligand stimulation increased the expression of mTOR in THP-1 cells., Conclusion: These novel findings on NOTCH signaling may contribute to the development of effective NOTCH-targeted therapies against leukemia.

Aim: To examine the effects of echinomycin, a compound that inhibits DNA-binding activity of hypoxia-inducible factor-1 (HIF1), on leukaemia cell growth., Materials and Methods: Three acute myeloid leukaemia cell lines and three T-lymphoblastic leukaemia cell lines were cultured with echinomycin. Cell growth, mRNA and protein expression levels were examined by WST-1 assay, reverse-transcription polymerase chain reaction and immunoblotting, respectively., Results: HIF1α protein was expressed in all cell lines under normoxia. Treatment with echinomycin suppressed cell growth and induced apoptosis in association with decreased mRNA expression of HIF1 targets, glucose transporter-1 (GLUT1) and B-cell CLL/lymphoma-2 (BCL2). Echinomycin also suppressed the protein expression of NOTCH1, cleaved NOTCH1, v-myc myelocytomatosis viral oncogene homolog (MYC), v-akt murine thymoma viral oncogene homolog-1 (AKT), phosphorylated AKT, mechanistic target of rapamycin (mTOR), and phosphorylated mTOR and increased that of cleaved caspase-3 in some cell lines., Conclusion: Echinomycin suppresses leukaemia cell growth in association with reduced NOTCH1 expression. This is the first report to show that HIF inhibitor treatment suppresses NOTCH1 signalling. HIF inhibitors could be novel candidates for a molecular-targeted therapy against leukaemia.

Aim: PP242 is a compound which inhibits both mammalian target of rapamycin complex-1 (mTORC1) and mTORC2. We examined the effects of PP242 and rapamycin on mTOR signalling and evaluated potential crosstalk with the NOTCH signalling in eight leukemia cell lines., Materials and Methods: We examined the effects of treatment with these inhibitors on cell growth and protein expression., Results: PP242 suppressed growth more potently than did rapamycin. In two cell lines poorly sensitive to PP242, PP242 failed to inhibit v-akt murine thymoma viral oncogene homolog (AKT) phosphorylation. Suppression of mTOR phosphorylation was weaker in myeloid cell lines. Rapamycin induced eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) hyperphosphorylation in three cell lines. Phosphorylation of both isoforms (p70 and p85) of S6 kinase (S6K) was suppressed in three cell lines; only p70 was suppressed in the others. NOTCH1 expression and activation were up-regulated by PP242 in one cell line but down-regulated in another., Conclusion: PP242 is a candidate for molecular-targeted leukemia therapy, although its effects must be evaluated on a case-by-case basis. Crosstalk was found between the mTOR and NOTCH signalling pathways.

Notch1 and its ligand Jagged1 are proteins with important roles in the growth of leukemia cells. Although the detection of Notch1 protein in acute lymphoblastic leukemia cells using immunoblot analysis has been previously reported, the expression patterns of Notch1 and Jagged1 detected by flow cytometry (FCM) in normal blood cells and various leukemia cells have not been well-characterised. In the present study, we examined the expression patterns of Notch1 and Jagged1 in 10 normal blood samples, 8 bone marrow samples, 11 leukemia/lymphoma cell lines and leukemia cells from 22 patients with acute myeloid leukemia (AML), mature T-cell neoplasms or B-cell chronic lymphocytic leukemia (B-CLL) using FCM. The results showed that Notch1 expression is relatively strong in monocytes and granulocytes but weak in lymphocytes. The expression of Notch1 is stronger in bone marrow cells than in the equivalent cells in blood. All the cell lines examined strongly expressed Notch1, and eight cell lines expressed Jagged1. In leukemia cells from patients, four AML samples expressed Notch1 and/or Jagged1. However, three samples expressed neither Notch1 and/or Jagged1 and none of the mature T-cell neoplasm samples expressed either protein. However, all B-CLL samples expressed high levels of both Notch1 and Jagged1. We found that the expression of Notch1 and Jagged1 is detected in various hematological malignancies by FCM. The examination of these proteins is likely to be useful in the characterisation of diseases and individual cases. Examination of these proteins may also be useful in the selection of patients most likely to benefit from novel molecular-targeted therapies using Notch inhibitors in the future.

The detection of a V617F mutation (G to T exchange at nucleotide 1,849) in the JAK2 gene is crucial for the diagnosis of myeloproliferative neoplasms (MPN) such as polycythemia vera. Although sequence analysis is the standard method for detection, it is not suitable for clinical examinations due to the requirement of expensive equipment. In this study, we evaluated the efficiencies of four PCR-based methods to detect JAK2 V617F: allele-specific PCR (AS-PCR), PCR-restriction fragment length polymorphism (PCR-RFLP), high-resolution melting analysis (HRM) and the quenching probe method (QP). The HEL cell line, which harbors a homozygous JAK2 V617F mutation, as well as bone marrow samples from 16 MPN patients and normal control samples, were used in this assessment. The sensitivity of the detection limit of all four methods was also examined using samples of HEL cells mixed in a variety of ratios with cells containing wild-type JAK2. The results of all four methods were found to be concordant. AS-PCR was shown to be the most sensitive; however, it produced false positive results. Although PCR-RFLP demonstrated high specificity, it was time consuming. By contrast, results were obtained using HRM and QP in only 2 h. It was easier to recognize the curves derived from the mutant allele obtained using QP. QP is also suitable for the rough estimation of allele burden. JAK2 V617F assays are mainly used for diagnosis at presentation in clinical settings. We therefore conclude that in situations where high sensitivity is not required, QP is the preferable method for the detection of JAK2 V617F. To the best of our knowledge, this is the first report to demonstrate the efficiency of the QP method for the detection of JAK2 V617F using a standard thermal cycler.

Background: Bone morphogenetic protein 4 (BMP4) signaling is involved in the maintenance of hematopoietic stem cells. However, the effects of BMP4 on leukemia and lymphoma cells are unknown., Materials and Methods: The effects of recombinant BMP4 on the in vitro growth of 12 leukemia and lymphoma cell lines were examined., Results: BMP4 treatment promoted the short-term growth of three cell lines and suppressed the growth of one. Induction of differentiation was not observed. BMP4 treatment suppressed the clonogenicity of four out of the six examined cell lines. BMP4 treatment promoted the growth of Jurkat cells but suppressed their ability to form colonies. BMP4 treatment up-regulated the phosphorylation of SMAD1/5/8 complex, indicating that BMP4 mediated signal transduction in the cells., Conclusion: BMP4 suppressed the clonogenicity of selected leukemia and lymphoma cell lines. The regulation of BMP4 signaling may be a useful therapeutic approach for leukemia and lymphoma, if appropriate cases are selected.

We report a case of sepsis caused by Rhodococcus corynebacterioides, identified using 16S rRNA gene sequencing, in a myelodysplastic syndrome patient who had undergone hematopoietic stem cell transplantation. This is the first report of R. corynebacterioides infection in a human.

To contribute to the innovation of the medical education system in Japan, we visited 35 medical schools and 5 institutes in 12 countries of North America, Europe, Australia and Asia in 2008-2010 and observed the education system. We met the deans, medical education committee and administration affairs and discussed about the desirable education system. We also observed the facilities of medical schools.Medical education system shows marked diversity in the world. There are three types of education course; non-graduate-entry program(non-GEP), graduate-entry program(GEP) and mixed program of non-GEP and GEP. Even in the same country, several types of medical schools coexist. Although the education methods are also various among medical schools, most of the medical schools have introduced tutorial system based on PBL or TBL and simulation-based learning to create excellent medical physicians. The medical education system is variable among countries depending on the social environment. Although the change in education program may not be necessary in Japan, we have to innovate education methods; clinical training by clinical clerkship must be made more developed to foster the training of the excellent clinical physicians, and tutorial education by PBL or TBL and simulation-based learning should be introduced more actively.

Background Aims: There is increasing interest in using γδ T cells (GDTC) for cancer immunotherapy. Most studies have been concerned with the Vδ2 subset in blood, for which several expansion protocols exist. We have developed a protocol to expand Vδ1 and Vδ2 preferentially from human blood. We have characterized these subsets and their specificities for leukemic targets., Methods: GDTC were isolated from the peripheral blood mononuclear cells (PBMC) of healthy donors via positive magnetic cell sorting; their proliferation in vitro was induced by exposure to the mitogen concanavalin A (Con A). CD107 and cytotoxicity (Cr(51)-release and flow cytometric) assays were performed. GDTC clones and target cells were immunophenotyped via flow cytometry., Results: Longer initial exposure to Con A typically resulted in higher Vδ1 prevalence. Vδ1 were activated by and cytotoxic to B-cell chronic lymphocytic leukemia (B-CLL)-derived MEC1 cells, whereas Vδ2 also responded to MEC1 but more so to the Philadelphia chromosome-positive [Ph+] leukemia cell line EM-enhanced green fluorescent protein (2eGFPluc). Vδ2 clone cytotoxicity against EM-2eGFPluc correlated with Vδ2 T-cell antigen receptor (TCR) and receptor found on Natural Killer cells and many T-cells (NKG2D), whereas Vδ1 clone cytotoxicity versus MEC1 correlated with Vδ1 TCR, CD56 and CD95 expression. Vδ1 also killed Epstein-Barr Virus (EBV)-negative B-CLL-derived TMD2 cells. Immunophenotyping revealed reduced HLA-ABC expression on EM-2eGFPluc, whereas MEC1 and TMD2 exhibited higher Tumor Necrosis Factor-Related Apoptosis-Inducing Ligand (TRAILR1)., Conclusions: Our ability to expand peripheral Vδ1 cells and show their cytotoxicity to B-CLL-derived cell lines suggests that this novel approach to the cellular treatment of B-CLL may be feasible.

Background: Hedgehog (Hh) signaling is involved in cancer cell growth. However, the effects of Hh stimulation on leukemia cells are unknown., Materials and Methods: The effects of recombinant sonic Hedgehog (Shh) protein on the in vitro growth of one B-lymphoma and four myeloid leukemia cell lines were examined., Results: Shh stimulation had no significant effect on the short-term growth of whole cell populations in any of the five cell lines. However, Shh promoted clonogenic cell recovery after suspension culture, suggesting promotion of leukemia stem or progenitor cell amplification in three cell lines. The lack of Hh receptors in one cell line and endogenous Shh expression in another were possible reasons for the lack of effects of Shh in these cases., Conclusion: These results suggest that Shh stimulation promotes the self-renewal capacity of leukemia stem cells in some cell lines. Inhibition of Hh signaling could represent a novel therapeutic approach in leukemia.