Takuya Matsunaga

Association of Higher Methotrexate Dose With Lymphoproliferative Disease Onset in Rheumatoid Arthritis Patients

Methotrexate (MTX) is used as an anchor drug for rheumatoid arthritis (RA). Lymphoproliferative disease (LPD) occasionally develops in patients treated with MTX, and is known as MTX‐associated LPD (MTX‐LPD). Although MTX‐LPD occurs mainly in RA patients, it has not been established if MTX administration is an independent risk factor for LPD in RA patients. We examined the clinical characteristics of MTX‐LPD in Japanese RA patients and attempted to determine the risk factors for MTX‐LPD development.

Elevated HIF-1α expression of acute myelogenous leukemia stem cells in the endosteal hypoxic zone may be a cause of minimal residual disease in bone marrow after chemotherapy

Acute myelogenous leukemia (AML) cells are generally hemosensitive, and 70–80% of AML patients undergo complete emission after chemotherapy [1]. However, long-term diseaseree survival remains as low as 30–50% [1], mainly because of elapse after chemotherapy. The relapse has been ascribed to minmal residual disease (MRD) in the bone marrow (BM) [2]. It has been hypothesized that a principal mechanism for MRD in he BM of AML is the presence of leukemic stem cells (LSCs), which re resistant to the cytotoxic effects of chemotherapy [3–6]. Expermental evidence suggests that the incorporation of LSCs in niches n the BM promotes the chemoresistance of AML [4–6]. Using a

Detection of EML4-ALK fusion genes in a few cancer cells from transbronchial cytological specimens utilizing immediate cytology during bronchoscopy

The presence of fusion genes between the anaplastic lymphoma kinase (ALK) and echinoderm microtubule-associated protein-like 4 (EML4) genes is useful for determining appropriate molecular-targeted therapies in patients with non-small cell lung cancer (NSCLC). The diagnosis of NSCLC is often judged from transbronchial cytological specimens. The efficacy of RT-PCR for detection of EML4-ALK fusion genes in transbronchial cytological specimens has not been studied. Here, we evaluated the detection rate of EML4-ALK fusion genes in transbronchial cytological specimens positive for NSCLC by immediate cytology during bronchoscopic examination. Various numbers of H2228 cells carrying EML4-ALK variant 3 were combined with 1×10(6) wild-type WBCs. The RNA was extracted and the sensitivity of detection of the EML4-ALK fusion gene was determined using a nested RT-PCR. A total of 161 cell samples, from cases without available tissue samples, obtained by bronchoscopic examinations utilized for immediate cytology in patients with NSCLC were subsequently analyzed for EML4-ALK fusion genes using a nested multiplex RT-PCR. EML4-ALK variant 3 was detected in a small number of H2228 cells (10 cells), even in the presence of 1×10(6) WBCs (sensitivity: 0.001%). In the patient cytological samples, EML4-ALK fusion genes were detected in five of 161 NSCLCs (3.1%) and four of 88 adenocarcinomas (4.5%). Sequencing confirmed that these samples included three variant 1 genes, one variant 2 gene and one variant 3 gene. Using the same cytological samples, EGFR mutations were detected in 39 of 161 NSCLCs (24.2%) and 36 of 88 adenocarcinomas (40.9%). There was no case in which both EML4-ALK fusion and EGFR mutation were simultaneously detected. Rapid diagnosis during bronchoscopy utilizing immediate cytology contributed to the selection of the best samples for genetic analysis. EML4-ALK fusion genes as well as EGFR mutations were successfully detected in a small number of cancer cells from transbronchial cytological specimens using a nested multiplex RT-PCR. Our present strategy can be integrated into the clinical process without additional invasive examination of patients. In the era of molecular-targeted treatments for NSCLC, the combination of rapid diagnosis during bronchoscopic examination and stocking samples as cDNA could further correspond to genetic analyses of accumulating driver genes in NSCLC.

Potentiated activation of VLA-4 and VLA-5 accelerates proplatelet-like formation

Fibronectin (FN) plays important roles in the proliferation, differentiation, and maintenance of megakaryocytic-lineage cells through FN receptors. However, substantial role of FN receptors and their functional assignment in proplatelet-like formation (PPF) of megakaryocytes are not yet fully understood. Herein, we investigated the effects of FN receptors on PPF using the CHRF-288 human megakaryoblastic cell line, which expresses VLA-4 and VLA-5 as FN receptors. FN and phorbol 12-myristate 13-acetate (PMA) were essential for inducing PPF in CHRF-288 cells. Blocking experiments using anti-β1-integrin monoclonal antibodies indicated that the adhesive interaction with FN via VLA-4 and VLA-5 were required for PPF. PPF induced by FN plus PMA was accelerated when CHRF-288 cells were enforced adhering to FN by TNIIIA2, a peptide derived from tenascin-C, which we recently found to induce β1-integrin activation. Adhesion to FN enhanced PMA-stimulated activation of extracellular signal-regulated protein kinase 1 (ERK1)/2 and enforced adhesion to FN via VLA-4 and VLA-5 by TNIIIA2-accelerated activation of ERK1/2 with FN plus PMA. However, c-Jun amino-terminal kinase 1 (JNK1), p38, and phosphoinositide-3 kinase (PI3K)/Akt were not stimulated by FN plus PMA, even with TNIIIA2. Thus, the enhanced activation of ERK1/2 by FN, PMA plus TNIIIA2 was responsible for acceleration of PPF with FN plus PMA.

Cytokeratins negatively regulate the invasive potential of lung cancer cell lines

Lung cancer cells express several cytokeratins (CKs) that are subdivided into type I (CK9-23) and type II (CK1-8) subclasses. The functions of CKs in lung cancer cells have not been fully elucidated. The purpose of this study was to investigate the role of CKs in the invasion of lung cancer cells. We investigated the expression levels of CK7, 8, 18 and 19 in 12 non-small cell lung cancer (NSCLC) and seven SCLC cell lines by quantitative immunoblotting. The expression levels of these four CKs were significantly higher in the NSCLC cells. The NSCLC cell line HI1017 expressed CK8 and 18; A549 cells expressed CK7, 8, 18 and 19, respectively. Invasive sublines of HI1017 and A549 were established by repeated selection of invasive cells using a membrane invasion chamber system. The invasive cell lines showed lower expression levels of CKs compared with the parental cells. Exogenous CK19 also resulted in a decrease in invasiveness of the HI1017 cells. Suppression of either CK8 or CK18 by short interfering RNAs led to a decrease in the total CKs and increased invasiveness of both the HI1017 and A549 cells. A549 cells expressed very low levels of CK19. Suppression of CK19 affected neither invasive ability nor total CK amount in the A549 cells. Our observations indicate that CK expression levels were inversely associated with invasiveness of the NSCLC cell lines, and suggest that expression levels of dominant CKs may affect invasive ability.

Membranous glomerulonephritis associated with Mycobacterium shimoidei pulmonary infection.

Patient: Male, 83 Final Diagnosis: Membranous glomerulonephritis Symptoms: Producting cough Medication: — Clinical Procedure: — Specialty: Nephrology Objective: Rare disease Background: Membranous glomerulonephritis can occur secondarily from infectious diseases. There are no reports describing membranous glomerulonephritis caused by non-tuberculous mycobacterium infection. However, several cases with membranous glomerulonephritis due to Mycobacterium tuberculosis have been reported. Mycobacterium shimoidei is an uncommon pathogen, and less than 20 cases with this species have been reported. A therapeutic regimen for this infection has not been established yet. Case Report: An 83-year-old Japanese man presented with productive cough for 6 months. Computed tomography scan showed multiple cavities in the bilateral pulmonary fields. Acid-fast bacilli were evident in his sputum by Ziehl-Neelsen staining (Gaffky 3). PCR amplifications for Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare were all negative. Finally, Mycobacterium shimoidei was identified by rpoB sequencing and 16S rRNA sequencing. Urine examination showed a sub-nephrotic range of proteinuria and histology of the kidney showed membranous glomerulonephritis. Antimycobacterial treatment with clarithromycin, rifampicin, and ethambutol dramatically improved not only the pulmonary disease, but also the proteinuria. Conclusions: To the best of our knowledge, the presented case is the first report showing non-tuberculous mycobacterium-induced secondary membranous glomerulonephritis. A combination with clarithromycin, ethambutol, and rifampicin might be effective for treatment of Mycobacterium shimoidei infection.

H2-receptor antagonist influences dasatinib pharmacokinetics in a patient with Philadelphia-positive acute lymphoblastic leukemia

We recently reported in this journal that administration of acid suppressant such as an H2-receptor antagonist (H2RA) and a proton pump inhibitor can decrease the absorption of dasatinib from the gastrointestinal tract, thereby resulting in a significant decrease in its plasma concentration. Here, we report a patient treated with dasatinib and H2RA famotidine for whom the total area under the total plasma concentration–time curve (AUC0–12) of dasatinib dramatically increased after cessation of famotidine from 505.7 to 1,816.3xa0ng·h/mL. This is the first report to confirm the drug interaction between dasatinib and H2RA by using sequential pharmacokinetic profiling.

A Novel Mechanism Underlying the Basic Defensive Response of Macrophages against Mycobacterium Infection

Following inhalation of Mycobacterium tuberculosis, including bacillus Calmette–Guérin (BCG), pathogens enter and grow inside macrophages by taking advantage of their phagocytic mechanisms. Macrophages often fail to eliminate intracellular M. tuberculosis, leading to the induction of host macrophage death. Despite accumulating evidence, the molecular mechanisms underlying M. tuberculosis infection–induced cell death remain controversial. In this study, we show the involvement of two distinct pathways triggered by TLR2 and β2 integrin in BCG infection–induced macrophage apoptosis. First, BCG infection induced activation of ERK1/2, which in turn caused phosphorylation/activation of the proapoptotic protein Bim in mouse macrophage-like Raw 264.7 cells. BCG-infected Raw cells treated with U0126, an MEK/ERK inhibitor, led to the suppression of Bim phosphorylation alongside a remarkable increase in the number of viable macrophages. Small interfering RNA–mediated knockdown of Bim rescued the macrophages from the apoptotic cell death induced by BCG infection. Stimulation with Pam3CSK, a TLR2 agonist, induced macrophage apoptosis with a concomitant increase in the phosphorylation/activation of MEK/ERK and Bim. These observations indicate the important role of the TLR2/MEK/ERK/Bim pathway in BCG infection–induced macrophage apoptosis. Second, we used the β2 integrin agonists C3bi and fibronectin to show that the β2 integrin–derived signal was involved in BCG infection–induced apoptosis, independent of MEK/ERK activation. Interestingly, latex beads coated with Pam3CSK and C3bi were able to induce apoptosis in macrophages to the same extent and specificity as that induced by BCG. Taken together, two distinct pattern-recognition membrane receptors, TLR2 and β2 integrin, acted as triggers in BCG infection–induced macrophage apoptosis, in which MEK/ERK activation played a crucial role following the engagement of TLR2.

Detection of Epidermal Growth Factor Receptor Mutations in a Few Cancer Cells from Transbronchial Cytologic Specimens by Reverse Transcriptase-Polymerase Chain Reaction

AbstractBackground: Epidermal growth factor receptor (EGFR) mutational status has the potential to be useful for determining prospective therapies in patients with non-small cell lung cancer (NSCLC) when analyzed in transbronchial cell specimens. The efficacy of RNA-based methods for the detection of EGFR mutations in transbronchial cell specimens has not been studied. Ultrafast Papanicolaou (UFP) staining is a method used in the immediate assessment of cytology during bronchoscopic examination.n Objectives: The aims of this study were (i) to compare the efficacy of RNA-based methodology for the detection of EGFR mutations with DNA-based methodology; and (ii) to assess the analysis of EGFR mutational status in transbronchial cell specimens, utilizing UFP staining.n Methods:EGFR mutant PC9 and NCI-H1975 cells were combined with wild-type EGFR white blood cells (WBCs), and the RNA and DNA were extracted. The sensitivity for the detection of EGFR mutations was determined. Polymerase chain reaction (PCR)-based methods, including reverse transcriptase (RT)-PCR and PCR-restriction fragment length polymorphism (RFLP), and sequencing were performed to detect the EGFR mutations. Seventy-one cell samples from bronchoscopic examinations that utilized UFP staining in patients with NSCLC were also analyzed for EGFR mutations.n Results:EGFR mutations were detected in a small number of cancer cells (ten cells), even in the presence of 1×106 WBCs, by the RNA-based methodology (either RT-PCR or PCR-RFLP) [sensitivity: <10−5]. However, the DNA-based method exhibited lower sensitivity (10−1). EGFR mutations were detected in 21 of 71 NSCLC samples (29.6%) and in 19 of 43 adenocarcinomas (44.2%) by the RNA-based methodology. The DNA-based methodology failed to detect EGFR mutations in several cases, while the RNA-based methodology was able to detect them.n Conclusions: Rapid diagnosis during bronchoscopy, utilizing UFP staining, contributed to the selection of the best samples for genetic analysis. EGFR mutations could be detected in a small number of cancer cells by the RNA-based methodology, with higher sensitivity than the DNA-based methodology, even in samples where numerous normal cells were present. Our present strategy can be integrated into the clinical process without additional invasive examination of patients and provides information regarding the EGFR mutational status.

Calmodulin antagonists induce cell cycle arrest and apoptosis in vitro and inhibit tumor growth in vivo in human multiple myeloma

BackgroundHuman multiple myeloma (MM) is an incurable hematological malignancy for which novel therapeutic agents are needed. Calmodulin (CaM) antagonists have been reported to induce apoptosis and inhibit tumor cell invasion and metastasis in various tumor models. However, the antitumor effects of CaM antagonists on MM are poorly understood. In this study, we investigated the antitumor effects of naphthalenesulfonamide derivative selective CaM antagonists W-7 and W-13 on MM cell lines both in vitro and in vivo.MethodsThe proliferative ability was analyzed by the WST-8 assay. Cell cycle was evaluated by flow cytometry after staining of cells with PI. Apoptosis was quantified by flow cytometry after double-staining of cells by Annexin-V/PI. Molecular changes of cell cycle and apoptosis were determined by Western blot. Intracellular calcium levels and mitochondrial membrane potentials were determined using Fluo-4/AM dye and JC-10 dye, respectively. Moreover, we examined the in vivo anti-MM effects of CaM antagonists using a murine xenograft model of the human MM cell line.ResultsTreatment with W-7 and W-13 resulted in the dose-dependent inhibition of cell proliferation in various MM cell lines. W-7 and W-13 induced G1 phase cell cycle arrest by downregulating cyclins and upregulating p21cip1. In addition, W-7 and W-13 induced apoptosis via caspase activation; this occurred partly through the elevation of intracellular calcium levels and mitochondrial membrane potential depolarization and through inhibition of the STAT3 phosphorylation and subsequent downregulation of Mcl-1 protein. In tumor xenograft mouse models, tumor growth rates in CaM antagonist-treated groups were significantly reduced compared with those in the vehicle-treated groups.ConclusionsOur results demonstrate that CaM antagonists induce cell cycle arrest, induce apoptosis via caspase activation, and inhibit tumor growth in a murine MM model and raise the possibility that inhibition of CaM might be a useful therapeutic strategy for the treatment of MM.