Purpose: Rituximab is commonly incorporated into CD20-positive B-cell lymphoma therapy to improve response and prognosis. With increasing use, resistance to rituximab is a continuing concern, but CD20 mutation as a cause of resistance has not previously been reported. Experimental Design: Freshly collected lymphoma cells from 50 patients with previously untreated or relapsed/resistant non-Hodgkin's B-cell lymphomas (diffuse large B cell, n = 22; follicular, n = 7; mucosa associated lymphoid tissue, n = 16; chronic lymphocytic leukemia, n = 2; small lymphocytic lymphoma, n = 1; lymphoplasmacytic, n = 1; mantle cell lymphoma, n = 1) were assessed for CD20 expression by flow cytometry, and CD20 gene sequencing was done on extracted DNA. Results: CD20 mutations were found in 11 (22.0%) of 50 patients and could be grouped as C-terminal deletion (8.0%), early termination (10.0%), and extracellular domain (2.0%) or transmembrane domain (2.0%) mutations. The mean fluorescence intensity of CD20 on fresh lymphoma cells was significantly lower for the C-terminal deletion mutation [3.26; 95% confidence interval (95% CI), 0.09-6.89] compared with wild type (30.8; 95% CI, 22.4-39.2; P < 0.05). In contrast, early termination mutations did not show significant differences in CD20 expression compared with wild type (19.5; 95% CI, 10.7-28.4; P > 0.05).Conclusions: It is possible that C-terminal deletion mutations of CD20 may be related to relapse/resistance after rituximab therapy. These mutations should be examined in patients showing progression of disease after partial remission. . 3), respectively. The rituximab target antigen is the B-cell membrane differentiation antigen CD20, and rituximab has emerged as a useful tool for adjunct cancer therapy (4). Although CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone/prednisolone) therapy leads to median overall survival rates of only 60%, addition of rituximab improves rates by f20% (5).With the need to determine standard first-, second-, and subsequent-line combination therapies using rituximab (6, 7), relapse/resistance to rituximab therapy is an important issue.The mechanisms of action of rituximab are inhibition of proliferation, induction of apoptosis, complement-dependent cytotoxicity, and antibody-dependent cellular cytotoxicity. A few reports indicate that loss of CD20 expression occurs in some patients with non-Hodgkin's lymphoma during rituximab therapy (8 -10), but the relationship between development of resistance to rituximab and changes in rituximab action have not yet been clarified. Heterogeneity of intensity of CD20 expression in replicate analysis of the same sample has been commonly observed by flow cytometric analysis (11). One explanation for this might be the development of resistant subsets of lymphoma cells by mutation. Recently, mutations in the epidermal growth factor receptor have been reported to have a relationship with the differing sensitivity to gefitinib therapy seen in samples from Japanese and American patients (12).Our ex...

Unusual physical characteristics of water can be easier explained and understood if properties of water clusters are revealed. Experimental investigation of water clusters has been reported by highly specialized equipment and/or harsh experimental conditions and has not determined the properties and the formation processes. In the current work, we used standard 1H-NMR as a versatile and facile tool to quantitatively investigate water clusters in the liquid phase under ambient conditions. This approach allows collection of data regarding the formation, long lifetime, stability, and physical properties of water clusters, as a cubic octamer in the liquid phase.

Recently, anti-CD20 (rituximab) and anti-Her2/neu (trastuzumab) antibodies have been developed and applied to the treatment of malignant lymphoma and breast cancer, respectively. However, bulky lymphoma is known to be resistant to rituximab therapy, and this needs to be overcome. Fresh lymphoma cells were collected from 30 patients with non-Hodgkin's lymphoma, the expression of CD20 and CD55 was examined by flow cytometry, and complement-dependent cytotoxicity (CDC) assays were carried out. Susceptibility to CDC with rituximab was decreased in a tumor size-dependent manner (r = -0.895, P < 0.0001), but not in a CD20-dependent manner (r = -0.076, P = 0.6807) using clinical samples. One complement-inhibitory protein, CD55, contributed to bulky lymphoma-related resistance to CDC with rituximab. A decrease in susceptibility to CDC with rituximab was statistically dependent on CD55 expression (r = -0.927, P < 0.0001) and the relationship between tumor size and CD55 expression showed a significant positive correlation (r = 0.921, P < 0.0001) using clinical samples. To overcome the resistance to rituximab by high expression of CD55 in bulky lymphoma masses, small interfering RNA (siRNA) was designed from the DNA sequence corresponding to nucleic acids 1-380 of the CD55 cDNA.

Edaravone (3-methyl-1-phenyl-2-pyrazolin-5-one) has been used as a free radical scavenging drug for the treatment of acute ischemic stroke in Japan since 2001. Edaravone is given to patients intravenously; therefore, it is distributed in the form of an aqueous solution. However, aqueous solutions of edaravone are very unstable because it is present as edaravone anion, which is capable of transferring an electron to free radicals including oxygen, and becomes edaravone radical. We observed the formation of hydrogen peroxide and edaravone trimer when aqueous edaravone solution was kept at 60°C for 4 weeks. We proposed the mechanism of edaravone trimer formation from edaravone radicals. Lowering the pH and deoxygenation can effectively increase the stability of aqueous edaravone solution, since the former reduces edaravone anion concentration and the latter inhibits edaravone radical formation. Addition of sodium bisulfite partially stabilized aqueous edaravone solutions and partially inhibited the formation of edaravone trimer. Formation of bisulfite adduct was suggested by 13C NMR and HPLC studies. Therefore, the stabilizing effect of sodium bisulfite is ascribed to the formation of a bisulfite adduct of edaravone and, consequently, reduction in the concentration of edaravone anion.

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Teaching materials characterization to support student research projects requires a systematic educational approach, because characterization involves a combination of analysis instruments. As analytical instruments are expensive, it is difficult to provide multiple sets simultaneously. An effective educational program allows students to select their own research materials to characterize and apply their personal strategies of instrumental analysis. These strategies are designed around the purposes of the analytical instruments, e.g., molecular structure analysis, crystal structure analysis, morphology assessment, surface analysis, elemental analysis, and thermal analysis. An open-ended laboratory complements this educational purpose. Here, we report on an open-ended laboratory program for fourth-year undergraduate and graduate students at the Materials Characterization Central Laboratory at Waseda University (Tokyo, Japan). The goals of our open-ended laboratory program are to enable students to (1) conduct instrumental analysis, (2) operate analytical instruments, and (3) interpret their data. A team led by a supervisor and laboratory staff offers students a flexible program. This flexibility can be applied to various research fields, such as macromolecular chemistry, inorganic chemistry, organic chemistry, physical chemistry, electrochemistry, physics, catalyst chemistry, biomaterials science, and chemical engineering. These diverse research fields demonstrate the feasibility of applying our open-ended laboratory program to student research projects.

Mass spectrometric differentiation of structural isomers is important for the analysis of forensic samples. Presently, there is no mass spectrometric method for differentiating halogen positional isomers of cannabimimetic compounds. We describe here a novel and practical method for differentiating one of these compounds, N-(1-amino-3-methyl-1-oxobutan-2-yl)-1-(4-fluorobenzyl)-1H-indazole-3-carboxamide (AB-FUBINACA (para)), and its fluoro positional (ortho and meta) isomers in the phenyl ring by electron ionization-triple quadrupole mass spectrometry. It was found that the three isomers differed in the relative abundance of the ion at m/z 109 and 253 in the product ion spectra, while the detected product ions were identical. The logarithmic values of the abundance ratio of the ions at m/z 109 to 253 (ln(A /A )) were in the order meta < ortho < para and increased linearly with collision energy. The differences in abundances were attributed to differences in the dissociation reactivity between the indazole moiety and the fluorobenzyl group because of the halogen-positional effect on the phenyl ring. Our methodology, which is based on the abundance of the product ions in mass spectra, should be applicable to determination of the structures of other newly encountered designer drugs. Copyright © 2016 John Wiley & Sons, Ltd.

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