Abstract Background Chronic lymphocytic leukemia (CLL) is the rarest adult leukemia in Japan, whereas it is the most common leukemia in the Western world. Recent studies from the United States and Germany suggest a possible etiological association between Merkel cell polyomavirus (MCPyV) and CLL, although no data have been reported from Eastern countries. To increase the volume of relevant data, this study investigated the prevalence and DNA loads of MCPyV and human polyomavirus 9 (HPyV9), another lymphotropic polyomavirus, in Japanese CLL cases. Findings We found that 9/27 CLL cases (33.3 %) were positive for MCPyV using quantitative real-time polymerase chain reaction analysis. The viral DNA loads ranged from 0.000017 to 0.0012 copies per cell. All cases were negative for HPyV9. One MCPyV-positive CLL case was evaluated by mutational analysis of the large T ( LT ) gene, which indicated the presence of wild-type MCPyV without a nucleotide deletion. DNA sequence analysis of the entire small T ( ST ) gene and the partial LT gene revealed that a Japanese MCPyV isolate, designated CLL-JK, had two nucleotide gaps when compared with the reference sequence of the North American isolate MCC350. Conclusions This study provides the first evidence that MCPyV is present in a subset of Japanese CLL cases with low viral DNA loads. MCPyV and HPyV9 are unlikely to contribute directly to the development of CLL in the majority of Japanese cases. MCPyV isolated from the Japanese CLL cases may constitute an Asian group and its pathogenicity needs to be clarified in future studies.
From 2001 to 2012, 71 individuals with hematological diseases received HSCT in our institution. Of these, 41 developed disseminated intravascular coagulation (DIC) in association with various underlying conditions. The patients who developed DIC after 2008 (n = 23) were treated by recombinant human soluble thrombomodulin (rTM), and the others (n = 11) were treated by either heparin and/or antithrombin III concentrate. Seven patients did not receive any anticoagulant therapy. Of note, treatment for coagulopathy by rTM significantly improved clinical outcomes of patients at day 100 and dramatically prolonged their overall survival (P = 0.044). Taken together, rTM is useful to improve clinical outcomes of transplant recipients with coagulopathy.
Globally, it is estimated that about 15% of all human malignancies are caused by viral infection [1]. These tumors characteristically have a long latent period between initial infection and subsequent malignancy, and most infected individuals never actually develop malignant disease. Multiple types of leukemia and lymphoma have been associated with preceding viral infection, including Epstein-Barr virus, human herpesvirus 8 (also called Kaposi sarcoma-associated herpesvirus) and human T-lymphotropic virus. These viruses establish their latency in lymphocytes and cause lymphoproliferative disorders.In 2008, a new human tumor virus designated 'Merkel cell polyomavirus' (MCPyV) was identified in Merkel cell carcinoma (MCC), a neuroendocrine carcinoma of the skin [2]. MCPyV has a close phylogenetic relationship to the African green monkey-derived lymphotropic polyomavirus and can infect human lymphoid cells. Since the presence of MCPyV DNA was demonstrated in some cases of lymphoproliferative disorders [3,4], the search for hematologic neoplasias in which MCPyV plays a role in the etiopathogenesis has become an important issue [5]. Among patients with lymphoproliferative disorders, patients with chronic lymphocytic leukemia (CLL), the most common leukemia of adults in the Western world, have higher MCPyV DNA detection rates [4]. Although most CLL tumors are not positive for MCPyV and the viral load is low, MCPyV may contribute to a significant proportion of CLL cases [6,7,8,9,10]. Our report is the first from the Eastern world to show the presence of MCPyV in a small number of Japanese CLL cases [8].In contrast to many studies searching for MCPyV in lymphoproliferative disorders, few studies have reported on the prevalence of MCPyV in myeloid disorders. Since it has been shown that MCPyV can also infect monocytic cells [11], there may be a role for MCPyV in disorders of the monocytic lineage [5]. If monocytes constitute a long-term reservoir of MCPyV, it is conceivable that this tumor virus may lead to the oncogenic transformation of the infected monocytes. In this context, we focused on monocytic leukemias to define another possible disease category related to MCPyV.Our study included leukemic specimens from 21 Japanese patients diagnosed with chronic myelomonocytic leukemia (CMML; 6 cases) and acute monocytic leukemia, including 5 M4 and 10 M5 cases according to the French-American-British (FAB) classification. The median ages of the patients were 78 years (range 64-84) for CMML, 60 years (range 45-92) for M4 and 66 years (range 16-83) for M5 cases. There were 4 male and 2 female CMML patients, 3 male and 2 female M4 patients, and 7 male and 3 female M5 patients. Total DNA was extracted using the standard phenol-chloroform method from a fraction containing abundant leukemia cells from peripheral bloods or bone marrow aspirates. This study was approved by the Ethics Committee of Kochi Medical School, Kochi University, Kochi, Japan.The extracted DNAs were subjected to quantitative TaqMan real-time polymerase chain reaction (PCR) analysis. The reaction was performed in triplicate on a StepOnePlus thermocycler (Life Technologies, Tokyo, Japan). Primers and probes were prepared to amplify the MCPyV small T-antigen gene (ST) and the human RNase P gene (RPP30). The primer sequences to detect the MCPyV LT gene were 5′-GCAAAAAAACTGTCTGACGTGG-3′ and 5′-CCACCAGTCAAAACTTTCCCA-3′, and the probe sequence was FAM-TATCAGTGCTTTATTCTTTGGTTTGGATTTCCTCCT-TAMRA. The primer sequences to detect the RPP30 gene were 5′-AGATTTGGACCTGCGAGCG-3′ and 5′-GAGCGGCTGTCTCCACAAGT-3′, and the probe sequence was FAM-TTCTGACCTGAAGGCTCTGCGCG-TAMRA. The reaction mixture was prepared in a total volume of 20 μl containing 100 ng of DNA, the TaqMan gene expression master mix (Life Technologies), 900 nM of each primer, and 250 nM dual-labeled probe. DNAs from 5 MCPyV-positive MCC tumors were used as the PCR-positive control. Water containing all the PCR components except the template DNA was used as the negative control. The reaction conditions were 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The viral DNA load was defined as the viral DNA copies per RPP30 gene copy, which represents the viral copy number per cell [7].Our real-time PCR assay consistently detected MCPyV DNA in the 5 MCC tumors as positive controls with a vial copy number of 3.73-5.01. However, MCPyV DNA sequences were not detected in any of our leukemia samples, suggesting that MCPyV is unlikely to have played a pathogenic role in the monocytic leukemias of these patients.CMML develops mainly in older individuals, with a median age of 65-75 years [12]. Although the etiology of CMML is unknown, some environmental agents have been suggested as risk factors for its development [12], highlighting the need for epidemiological research on CMML. Acute myelomonocytic leukemia (FAB M4) occurs in all age groups but is more common in older individuals. Acute monoblastic and monocytic leukemia (FAB M5) may occur at any age but is common in young individuals. However, risk factors for these acute monocytic leukemias have not been defined. In this study, we searched for a viral etiology for leukemia of the monocytic lineage by focusing on MCPyV under the following hypothesis. The long-term MCPyV-persistent infection in monocytes may allow the virus to integrate its viral genome into the host cell genome, where it participates in the cell transformation process. This oncogenic process, together with the immunosuppression of the host or loss of specific surveillance for MCPyV epitopes and additional cellular changes or mutations, is a well-known multistep cell transformation mechanism, as shown for other tumor viruses. Although we failed to detect MCPyV DNA in the neoplastic monocytes using the highly sensitive real-time PCR assay, we have provided the first data on the prevalence of MCPyV in monocytic leukemias. Further worldwide epidemiological surveys are warranted to determine the possible association between MCPyV and disorders of the monocytic lineage.This study was supported financially by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Science and Technology of Japan.
Loss of function in lysosomal arylsulfatase B (ARSB) leads to Maroteaux–Lamy syndrome, a form of the mucopolysaccharidoses. Mutations in ARSB, at least those characterized in detail, often destabilize and interfere with the folding of the ARSB protein, resulting in the loss of functional ARSB in lysosomes. Pharmacological chaperones, which are ligands that assist in protein folding by binding to folding intermediates in the endoplasmic reticulum, are proposed to be potential drug candidates for such protein misfolding diseases. However, small-molecule ligands for ARSB have not been widely studied and most of the known ligands are sulfate compounds, which are highly polar and do not readily cross the membrane. Since pharmacological chaperones must be able to enter the cell and the endoplasmic reticulum, a surrogate pharmacophore with membrane permeable properties is needed. In this study, we identified phenylboronic acid as an effective sulfate surrogate with membrane permeability, via competitive enzymatic assay against ARSB. Additionally, phenylboronic acids were more potent at neutral pH and less so at acidic pH, exhibiting a pH selective activity profile ideal for pharmacological chaperones. Subsequent structure-activity relationship studies identified more potent derivatives, and ARSB was protected from thermal denaturation in the presence of the derivatives, supporting direct binding of the phenylboronic acids. Although further studies will be required to determine if these phenylboronic acids could act as pharmacological chaperones for ARSB mutants, our finding of phenylboronic acid as a pH-selective surrogate pharmacophore for the aryl sulfate should be valuable for designing pharmacological chaperones for sulfatases in the future.
// Hiroaki Kikuchi 1, 2 , Tomonori Higuchi 1 , Yumiko Hashida 1 , Ayuko Taniguchi 3 , Mikio Kamioka 4 , Takahiro Taguchi 5 , Akihito Yokoyama 3 , Ichiro Murakami 6 , Mikiya Fujieda 2 and Masanori Daibata 1 1 Department of Microbiology and Infection, Kochi Medical School, Kochi University, Nankoku, Kochi 783-8505, Japan 2 Department of Pediatrics, Kochi Medical School, Kochi University, Nankoku, Kochi 783-8505, Japan 3 Department of Hematology and Respiratory Medicine, Kochi Medical School, Kochi University, Nankoku, Kochi 783-8505, Japan 4 Department of Laboratory Medicine, Kochi Medical School, Kochi University, Nankoku, Kochi 783-8505, Japan 5 Department of Molecular and Cellular Biology, Kochi Medical School, Kochi University, Nankoku, Kochi 783-8505, Japan 6 Department of Pathology, Kochi Medical School, Kochi University, Nankoku, Kochi 783-8505, Japan Correspondence to: Masanori Daibata, email: daibatam@kochi-u.ac.jp Keywords: double hit lymphoma; MYC; BCL6; PLK1; HDAC Received: June 21, 2018 Accepted: August 10, 2018 Published: September 11, 2018 ABSTRACT "Double-hit" lymphoma (DHL) is a high-grade B-cell lymphoma that harbors concurrent MYC and BCL2 or BCL6 rearrangements. Because cases of MYC / BCL6 DHL are uncommon, most reported conclusions have been based on cases of MYC / BCL2 DHL. Lack of experimental MYC / BCL6 DHL models continues to hinder the pathophysiologic and therapeutic investigations of this disorder. We herein describe a novel MYC / BCL6 DHL cell line, designated DH-My6, carrying both the MYC – IGH and BCL6 – IGH fusion genes. Interruptions of MYC and BCL6 expressions using short interfering RNAs and chemical inhibitors led to significant attenuation of DH-My6 cell growth. Greater antitumor effects were found when the cells were treated with a combination of MYC and BCL6 inhibitors. Moreover, the PLK1 inhibitor volasertib and the HDAC inhibitor vorinostat synergized strongly when combined with the bromodomain inhibitor JQ1. DH-My6 is a new well-validated MYC / BCL6 DHL cell line that will provide a useful model for studies of the pathogenesis and therapeutics for the less common DHL tumor type. The rationale for approaches targeting both MYC and BCL6, and in combination with PLK1 or HDAC inhibitors for superior suppression of the aggressive MYC / BCL6 DHL warrants further in vivo testing in a preclinical model.
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