We previously evaluated Wilms’ tumor gene 1 (WT1) peptide vaccination in a large number of patients with leukemia or solid tumors and have reported that HLA‐A*24:02 restricted, 9‐mer WT1‐235 peptide (CYTWNQMNL) vaccine induces cellular immune responses and elicits WT1‐235‐specific cytotoxic T lymphocytes (CTLs). However, whether this vaccine induces humoral immune responses to produce WT1 antibody remains unknown. Thus, we measured IgG antibody levels against the WT1‐235 peptide (WT1‐235 IgG antibody) in patients with glioblastoma multiforme (GBM) receiving the WT1 peptide vaccine. The WT1‐235 IgG antibody, which was undetectable before vaccination, became detectable in 30 (50.8%) of a total of 59 patients during 3 months of WT1 peptide vaccination. The dominant WT1‐235 IgG antibody subclass was Th1‐type, IgG1 and IgG3. WT1‐235 IgG antibody production was significantly and positively correlated with both progression‐free survival (PFS) and overall survival (OS). Importantly, the combination of WT1‐235 IgG antibody production and positive delayed type‐hypersensitivity (DTH) to the WT1‐235 peptide was a better prognostic marker for long‐term OS than either parameter alone. These results suggested that WT1‐235 peptide vaccination induces not only WT1‐235‐specific CTLs as previously described but also WT1‐235‐specific humoral immune responses associated with antitumor cellular immune response. Our results indicate that the WT1 IgG antibody against the WT1 peptide may be a useful predictive marker, with better predictive performance in combination with DTH to WT1 peptide, and provide a new insight into the antitumor immune response induction in WT1 peptide vaccine‐treated patients.

In this study, we investigated the properties of monkey liver aldehyde oxidase directed toward the clarification of species differences. The aldehyde oxidase preparation purified from male cynomolgus monkey liver cytosol showed a major 150 kDa Coomassie brilliant blue (CBB)-stained band together with a minor 130 kDa band using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Both bands were identified as being aldehyde oxidase by a database search of the MS data obtained with nano-liquid chromatography, quardrupole time of flight, mass spectrometry (nano-LC Q/TOF MS). Based on the sequence coverage, the 130 kDa protein was presumed to be deficient in 20-30 kDa mass from the N-terminus. Full male cynomolgus monkey aldehyde oxidase cDNA was cloned and sequenced with the four degenerate primers designed by considering the peptide sequences containing the amino acids specific for monkey aldehyde oxidase. The deduced amino acid sequences had 96% amino acid identity with those of human enzyme. The aldehyde oxidase expressed in Escherichia coli also exhibited two immunoreactive bands on SDS-PAGE/Western blot analysis. Further, the biphasic pattern was observed for Eadie-Hofstee plots of the (S)-enantiospecific 2-oxidation activity of RS-8359 with the expressed and cytosolic monkey liver aldehyde oxidase. The results suggested that two forms of aldehyde oxidase in monkey were the expression products by a single gene. In contrast, the similarly expressed rat aldehyde oxidase showed only one immunoreactive protein and monophasic pattern. The biphasic phenomenon could be caused by the existence of two aldehyde oxidase isoforms or two active sites in a single enzyme or some other reasons. Further studies on the problems of the biphasic pattern and species differences in aldehyde oxidase are needed.

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Amine oxidases (AOs) are widely distributed among all living organisms. 1) This class of enzymes is divided into two subclasses: FAD-containing amine oxidases (FAD-AOs) and Cu-containing amine oxidases (Cu-AOs). Two types of mitochondrial monoamine oxidases, monoamine oxidases (MAO)-A and MAO-B, and cytosolic polyamine oxidase are included in FAD-AOs.2) The Cu-AOs consist of retina amine oxidase, diamine oxidase (DAO), and semicarbazide-sensitive amine oxidase (SSAO), which is known as the tissue-bound or plasma form of amine oxidase. The classification of MAO into an A and B form was primarily based on Johnston's presentation that both enzymes could be differentiated with the aid of the specific inhibition of MAO-A by clorgyline and of MAO-B by deprenyl. 3) Classification by means of specific inhibitors has been more convenient for the identification of different types of amine oxidase. However, this concept has been considered somewhat of an oversimplification and the premises on which it was based have come under increasing scrutiny. [4][5][6][7][8][9] One major problem was that serotonin and b-phenylethylamine, which are generally used to distinguish MAO-A and MAO-B, are not entirely specific for the respective MAO form. Another problem was that the substrate selectivity of MAO seems to be affected by minor cross-inhibition caused by specific inhibitors.SSAO essentially metabolizes amine compounds as well as MAO-A and MAO-B. However, the enzyme activity is not inhibited by clorgyline and deprenyl, whereas it is almost completely inhibited by semicarbazide. In contrast, semicarbazide has a weak inhibitory effect on MAO at a concentration of 0.1-1 mM. 10) Clorgyline and deprenyl have been regarded as virtually inactive against the tissue-bound SSAO, nevertheless they showed moderate reversible competitive inhibition against bovine 11) and human 12,13) plasma SSAO at a concentration of 0.1-1 mM. DAO, belonging to the Cu-AOs, is responsible for histamine metabolism. 14) Comparative substrate selectivity studies of DAO have not always been performed in detail. Aminoguanidine has been used for many years as an inhibitor of Cu-AO, but it is a relatively weak and nonselective inhibitor of SSAO. 15) Thus, the results of the substrate selectivity of these amine oxidases using specific inhibitors are very confused and complex, and might not reflect natural biochemical properties. In this study, we utilized a single amine oxidase expressed in COS-1 cells to investigate the substrate selectivity of amine oxidases in the absence of chemical inhibitors. MATERIALS AND METHODSAnimals Nine-week-old male Wistar rats purchased from Japan SLC (200-220 g, Hamamatsu, Japan) were housed at a constant temperature (23Ϯ1°C) and humidity (55Ϯ5%) with automatically controlled lighting (07:00-19:00).Plasmid Construction Standard protocols were used for all recombinant DNA technology. 16) The full-length ORF AOs plasmid was constructed as follows. Total RNA was isolated using the Wizard SV Total RNA Extraction Kit (Promega, Madison, WI,...

Aldehyde oxidase (AO) is a homodimer with a molecular weight of 300 kDa. To clarify the reasons for the well-known differences in rat strains, we set out to study the relationship between AO activity and the expression levels of its dimer. AO-catalyzed 2-oxidation activity of (S)-RS-8359 was measured in liver cytosols from ten rat strains. The expression levels of AO dimeric protein were evaluated by the native-PAGE/Western blot. Rat strains with low AO activity showed only a monomer, whereas strains with high activity overwhelmingly exhibited a dimer. Exceptionally, one strain in the high AO activity group displayed complex mixed expression patterns of low and high AO activity groups. However, there was a good relationship between AO activity and the expression levels of a dimer, but not of a monomer. The results suggest that rat strains with low AO activity lack the ability to produce a dimer necessary for catalytic activity, and AO differences in rat strains should be discussed in terms of the expression levels of the dimer itself.