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Human SSX was first identified as the gene involved in the t(X;18) translocation in synovial sarcoma. SSX is a multigene family, with 9 complete genes on chromosome Xp11. Normally expressed almost exclusively in testis, SSX mRNA is expressed in various human tumors, defining SSX as a cancer/testis antigen. We have now cloned the mouse ortholog of SSX. Mouse SSX genes can be divided into Ssxa and Ssxb subfamilies based on sequence homology. Ssxa has only one member, whereas 12 Ssxb genes, Ssxb1 to Ssxb12, were identified by cDNA cloning from mouse testis and mouse tumors. Both Ssxa and Ssxb are located on chromosome X and show tissue-restricted mRNA expression to testis among normal tissues. All putative human and mouse SSX proteins share conserved KRAB and SSX-RD domains. Mouse tumors were found to express some, but not all, Ssxb genes, similar to the SSX activation in human tumors. [Copyright &y& Elsevier]

We report on the effects of postdeposition annealing ambient on the hysteresis observed in the C-V measurement of Al2O3/GeO2 gate-dielectric stacks fabricated on Ge substrates. The results indicate that two types of oxide trap are responsible for the observed hysteresis: a type-I oxide trap that causes persistent C-V hysteresis and a type-II oxide trap that disappears when gate voltage is biased once in the accumulation region. We show that both types of oxide trap reside in the capacitor annealed in O2 ambient, but that only the type-II oxide trap resides in the capacitor annealed in N2 + 10% H2. Time-domain measurements of absorption current suggest that holes injected into the gate-dielectric stack induce the electronic deactivation of the type-II oxide trap. [ABSTRACT FROM AUTHOR]

Hydroxyapatite (HAP) is a main mineral constituent of bone and tooth and has an outstanding biocompatibility. HAP is a possible sorbent for heavy metals in wastewater due to its high adsorption capacity and low water solubility. We developed a removal system of Sr from aqueous solution by HAP column procedure. More than 90 % of Sr was adsorbed and removed from the Sr containing solution. Divalent cations, Ca, had little effect on the removal of Sr up to a concentration of 1 mmol L. This clearly indicates that the HAP column technique is advantageous with respect to the capacity to adsorb Sr from water present in the environment. [ABSTRACT FROM AUTHOR]

Presents a study which proposed the Carry and power assist robot for Unspecified Landform (CUL) walking robot to assist in rescue operations. Use of the Following Mode mechanism to enable the rescue robot CUL to walk in disaster areas; Details on the proposed concrete method called Mold Tube Method to realize the Following Mode concept; Details on the design of a prototype rescue robot CUL.

One year after the deposition of radionuclides from the Fukushima 1 Nuclear Power Plant (A formal name is Fukushima Daiichi Nuclear Power Station) in March 2011, radiocesium (Cs, Cs) concentrations ([Cs]) were comprehensively investigated in the wild plants of 99 species most of which were annual or summer green perennial herbs and started to grow from April 2012 at the heavily contaminated fields of paddy (three study sites) and upland (one study site) in Fukushima Prefecture. The survey was conducted three times (April, July and October) in the year. In each site, soils (soil cores of 5-cm depth) and plants (aerial shoots) were collected for determination of [Cs] on a dry weight basis, and then the transfer factor (TF) of radiocesium from soil to plant ([Cs]/[Cs]) was estimated in each species. The [Cs] values of both soils and plants largely varied. However, some species exhibited relatively high TF values (more than 0.4) (e.g., Athyrium yokoscense, Dryopteris tokyoensis, and Cyperus brevifolius), while others exhibited almost negligible values (less than 0.01) (e.g., Salix miyabeana, Humulus scandens, and Elymus tsukushiensis). In addition, judging from the 11 species grown in both paddy and upland fields, TF values were generally higher in the paddy fields. The estimation of phytoextraction efficiency of soil radiocesium by weed communities in the paddy fields suggests that the weed community is not a practical candidate for phytoremediation technique. [ABSTRACT FROM AUTHOR]

Ion exchange is a simple and efficient method for separating no-carrier-added Cu from an irradiated Ni target. We developed a semi-automated two-round Cu separation system equipped with a strong-base anion exchange resin column. We first verified the efficiency of the system using a non-radioactive substitute consisting of 25 mg of Ni and 127 ng of Cu, and confirmed that Cu was completely eluted at the second round of the separation step. After the bombardment, separation of Cu from the Ni target was achieved with high radiochemical purity. Cu produced and separated in this study had an extremely low level of Ni impurity. It could be used for labeling monoclonal antibodies for antibody positron emission tomography imaging and synthesizing radiopharmaceuticals. [ABSTRACT FROM AUTHOR]

Background: Identification of new cancer antigens is necessary for the efficient diagnosis and immunotherapy. A variety of tumor antigens have been identified by several methodologies. Among those antigens, cancer/testis (CT) antigens have became promising targets. Methods: The serological identification of antigens by the recombinant expression cloning (SEREX) methodology has been successfully used for the identification of cancer/testis (CT) antigens. We performed the SEREX analysis of colon cancer. Results: We isolated a total of 60 positive cDNA clones comprising 38 different genes. They included 2 genes with testis-specific expression profiles in the UniGene database, such as TEKT5 and a CT-like gene, A kinase anchoring protein 3 (AKAP3). Quantitative real-time RT-PCR analysis showed that the expression of TEKT5 was restricted to the testis in normal adult tissues. In malignant tissues, TEKT5 was aberrantly expressed in a variety of cancers, including colon cancer. A serological survey of 101 cancer patients with different cancers by ELISA revealed antibodies to TEKT5 in 13 patients, including colon cancer. None of the 16 healthy donor serum samples were reactive in the same test. Conclusion: We identified candidate new CT antigen of colon cancer, TEKT5. The findings indicate that TEKT5 is immunogenic in humans, and suggest its potential use as diagnostic as well as an immunotherapeutic reagent for cancer patients. [ABSTRACT FROM AUTHOR]

Abstract: Serological analysis of a recombinant cDNA expression library (SEREX) derived from two lung adenocarcinoma cancer cell lines using autologous sera led to the isolation of 41 positive cDNA clones comprising 28 different antigens. They coded for a variety of nuclear and cytoplasmic proteins. Among the antigens, nucleoporin 107 (NUP107) was isolated most frequently (5 of 41 clones). The second most frequently isolated antigen was coded for by C21orf58 (4 of 41 clones). During serological analysis of selected antigens based on their reactivity to sera from normal individuals and lung cancer patients, none of the antigens showed a cancer-restricted recognition pattern. However, five genes including NUP107 showed higher expression when we examined the changes in gene expression in five different adenocarcinoma cell lines, including those used in SEREX, compared with their levels in normal lung tissues by cDNA microarray analysis. On the other hand, the expression levels of five genes including C21orf58 were down regulated in all adenocarcinoma cell lines. This SEREX study combining comprehensive gene expression assays has added to the growing list of lung cancer antigens, which may aid the development of diagnostic and immunotherapeutic reagents for patients with lung cancer. [ABSTRACT FROM AUTHOR]

Abstract: We have investigated the electrical characteristics of tantalum oxy-nitride (TaON) films deposited using electron cyclotron resonance (ECR) plasma sputtering. A pure tantalum metal target was used as raw material combined with gases of oxygen and nitrogen. The electrical properties have been measured using metal–insulator–metal (MIM) structures of Al/TaON/Ru/Si or metal–insulator–semiconductor (MIS) structure of Al/TaON/Si. By controlling the oxygen gas flow in a moderate low gas flow rate at fixed nitrogen gas flow, TaON films have been stably obtained with the refractive indices of over 2.5 at 632.8nm wavelength, and the dielectric constants of over 30. However, the leakage currents have increased with an increase in the dielectric constants. To improve the leakage current, we have investigated the periodical deposition process, in which the ECR plasma irradiation was additionally introduced after thin TaON film was deposited. The breakdown strength of about 1MV/cm was obtained by the measurement using MIM structure. By the estimation of the C–V characteristics of the silicon–MIS structure using TaON, the dielectric constant of 34 was obtained for a TaON thickness of 12nm. [Copyright &y& Elsevier]

In this paper, we present an equivalent circuit model of a germanium (Ge) MIS structure that is biased in the inversion region, which includes the effects of the high intrinsic carrier density and high diffusion-limited conductance of the Ge substrate at room temperature. The model can successfully express the gate bias and frequency dependences of the capacitance characteristics that are specific to the Ge MIS capacitor. Moreover, it will be shown that the interface trap density and its gate bias dependence in the inversion region can be spectroscopically determined from the gate bias and measurement frequency dependences of the equivalent parallel conductance of the Ge surface. [ABSTRACT FROM AUTHOR]

Background and Aim: The present study was designed to investigate the expression of and humoral response against NY-ESO-1 in patients with hepatocellular carcinoma and to analyze the relationship between expression of NY-ESO-1 mRNA and clinicopathological features. Methods: NY-ESO-1 mRNA and protein expression in surgically resected hepatocellular carcinoma specimens, adjacent non-cancerous liver and non-tumor bearing liver were examined by reverse transcription-polymerase chain reaction and immunohistochemical staining using a monoclonal antibody against NY-ESO-1 (ES121), respectively. The antibody response to NY-ESO-1 was examined by enzyme-linked immunosorbent assay using recombinant NY-ESO-1 protein. Results: NY-ESO-1 mRNA was detected in 18 of 41 (43.9%) hepatocellular carcinomas. No NY-ESO-1 mRNA was expressed in 41 paired non-cancerous specimens and 18 specimens histologically diagnosed as liver cirrhosis or chronic hepatitis. Immunohistochemistry revealed heterogeneous expression of NY-ESO-1 protein in three of 18 NY-ESO-1 mRNA-positive hepatocellular carcinomas. None of 23 NY-ESO-1 mRNA-negative hepatocellular carcinomas expressed NY-ESO-1 protein. Antibody against NY-ESO-1 protein was detected in two of 92 patients with hepatocellular carcinoma. Both of these patients had tumors invading main branches of the portal vein. Conclusions: The present study has demonstrated the expression of NY-ESO-1 mRNA in hepatocellular carcinoma and NY-ESO-1 antibody production in patients with advanced hepatocellular carcinoma. Although the enhancement of NY-ESO-1 protein expression and the activation of immune response of the patients with hepatocellular carcinoma are necessary, NY-ESO-1 has the potential to be a good target molecule for immunotherapy against advanced hepatocellular carcinoma. [ABSTRACT FROM AUTHOR]

Background: Insulin-like growth factor binding protein (IGFBP)-3 functions as a carrier of insulin-like growth factors (IGFs) in circulation and a mediator of the growth suppression signal in cells. There are two reported p53 regulatory regions in the IGFBP3 gene; one upstream of the promoter and one intronic. We previously reported a hot spot of promoter hypermethylation of IGFBP-3 in human hepatocellular carcinomas and derivative cell lines. As the hot spot locates at the putative upstream p53 consensus sequences, these p53 consensus sequences are really functional is a question to be answered. Methods: In this study, we examined the p53 consensus sequences upstream of the IGFBP-3 promoter for the p53 induced expression of IGFBP-3. Deletion, mutagenesis, and methylation constructs of IGFBP-3 promoter were assessed in the human hepatoblastoma cell line HepG2 for promoter activity. Results: Deletions and mutations of these sequences completely abolished the expression of IGFBP-3 in the presence of p53 overexpression. In vitro methylation of these p53 consensus sequences also suppressed IGFBP-3 expression. In contrast, the expression of IGFBP-3 was not affected in the absence of p53 overexpression. Further, we observed by electrophoresis mobility shift assay that p53 binding to the promoter region was diminished when methylated. Conclusion: From these observations, we conclude that four out of eleven p53 consensus sequences upstream of the IGFBP-3 promoter are essential for the p53 induced expression of IGFBP-3, and hypermethylation of these sequences selectively suppresses p53 induced IGFBP-3 expression in HepG2 cells. [ABSTRACT FROM AUTHOR]

We have investigated the effects of the formation temperature and postmetallization annealing (PMA) on the interface properties of GeNx/p-Ge fabricated by the plasma nitridation of Ge substrates using an electron-cyclotron-resonance-generated nitrogen plasma. The nitridation temperature is found to be a critical parameter in improving the finally obtained GeNx/Ge interface properties. The GeNx/Ge formed at room temperature and treated by PMA at 400 °C exhibits the best interface properties with an interface trap density of 1 × 1011 cm-2 eV-1. The GeNx/Ge interface is unpinned and the Fermi level at the Ge surface can move from the valence band edge to the conduction band edge. [ABSTRACT FROM AUTHOR]

We have investigated GeNx/Ge interface properties using Si3N4(7 nm)/GeNx(2 nm)/Ge metal-insulator-semiconductor structures fabricated by the plasma nitridation of Ge substrates using an electron-cyclotron-resonance-generated nitrogen plasma. The interface trap density (Dit) measured by the conductance method is found to be distributed symmetrically in the Ge band gap with a minimum Dit value lower than 3 × 1011 cm-2eV-1 near the midgap. This result may lead to the development of processes for the fabrication of p- and n-Ge Schottky-barrier (SB) source/drain metal-insulator-semiconductor field-effect transistors using chemically and thermally robust GeNx dielectrics as interlayers for SB source/drain contacts and high-κ gate dielectrics. [ABSTRACT FROM AUTHOR]

The purpose of this study was to conduct a self-reported questionnaire survey of work-related musculoskeletal disorders (WMSDs) among Japanese radiological technologists (RTs) and to report on the relationship between wearing a lead apron and WMSDs. Between February and April of 2021, RTs in Okayama Prefecture, Japan, were surveyed by mail and through a website. Information on individual characteristics, physical factors at work, and the presence of WMSDs were collected. All participants were also asked whether they frequently wore lead aprons. A multiple logistic regression analysis was used to assess the relationship between wearing a lead apron and WMSDs. The model was adjusted for age, sex, body mass index (BMI), and working hours. Of the 123 participants, 67 (54.5%) had WMSDs. Multiple logistic regression analysis revealed that WMSDs were significantly associated with wearing a lead apron. Compared to the "Never wear" group, the odds ratios for the "Always/Frequently wear" and "Sometimes/Rarely wear" groups were 7.87 (95% confidence interval [CI]=1.28-48.46; p=0.026) and 7.80 (95% CI=1.43-42.44; p=0.017), respectively. Our analysis suggests that wearing a lead apron is associated with WMSDs, and thus design modifications in lead aprons may improve the occupational health management of RTs., Competing Interests: No potential conflict of interest relevant to this article was reported.

Anticancer drugs induce cell-cycle arrest and apoptosis not only in tumor cells, but also in immune cells. However, many preclinical and clinical findings show that some chemotherapeutic agents can improve the antitumor efficacy of immunotherapy. We immunohistochemically analyzed the degree of immune cell infiltration and the relevance of programmed cell death 1 ligand-1 (PD-L1) expression in surgically resected oral squamous cell carcinoma (OSCC) specimens from patients who had undergone pretreatment with certain chemotherapies and other patients without pretreatment. We divided the patients into the group of neoadjuvant chemotherapy (NAC) patients (n=8) and the nNAC (without NAC) patient group (n=10). We observed that NAC induced infiltrations of CD4, CD8 T cells and CD56 NK cells into the tumor microenvironment. Decreased numbers of Tregs and PD-1-positive cells were observed in the NAC group. No significant difference was observed in the degree of immune-cell infiltration between the patient groups except for CD56 NK cells in the stroma and PD-1 cells in cancer nests. Eighty percent of the nNAC specimens showed intermediate-to-strong PD-L1 protein expression, whereas 75% of the NAC specimens showed down-regulation of the PD-L1 protein, indicating the effectiveness of the chemotherapeutic treatment before surgery., Competing Interests: No potential conflict of interest relevant to this article was reported.

No research has been conducted on the radiation influence of tobacco on the alimentary system, although there have been some previous works on the respiratory system. In this study, the radioactive concentrations of 210Pb and 40K in a cigarette sample were first measured. The transfer factors of the nuclides from tobacco into smoke and solution (saliva and/or alcohol) were then examined. Moreover, the radiation doses from smoke inhalation were also evaluated. The radioactive concentrations of 210Pb and 40K in the cigarette tobacco were 0.01 and 0.3 Bq/cigarette. Since this 210Pb activity and the 210Po activity previously reported for the same sample were comparable, it can be concluded that there was a radioactive equilibrium between the 2 nuclides. The observed transfer factor of 210Pb (12%) into smoke was almost the same as that of 40K (15%), whereas the reported value for 210Po (60%) was significantly higher. The radiation doses due to inhalation of cigarette smoke varied from organ to organ, depending on the organotropic properties of the nuclide. For example, the kidneys, respiratory tract, and spleen showed relatively high doses from 210Pb and 210Po. The leaching rates indicated an inconsistent tendency related to solution types. This result could suggest that alcohol drinking, which is common in smokers, does not especially enhance the leaching characteristics.

Pulmonary surfactant has been used as a carrier to deliver a therapeutic virus to dysfunctional lung cells that reside within an intricate lung structure. To investigate whether pulmonary surfactant enhances the efficacy of intratracheal instillation of a therapeutic virus to target KRAS mutation-bearing lung cancer in vivo, we developed a recombinant adenovirus that induces cell death only in lung cancer cells and injected the adenovirus into a mouse model of KRAS mutation-positive lung cancer intratracheally with and without surfactant. A therapeutic adenovirus that induces cell death only in lung cancer cells was constructed by combining a cancer-specific human telomerase reverse transcriptase (hTERT) promoter fused to CCAAT/enhancer-binding protein alpha (CEBPα) with a modified lung-specific Clara cell-specific 10-kDa protein (CC10) promoter fused to cytotoxic adenovirus type 5 early region 1A (E1A). CEBPα is induced only in cancer cells and activates the CC10 promoter, which in turn induces cytotoxic E1A, and causes cell death only in lung cancer cells in vitro. This adenovirus was intratracheally administered to the model mice (CCSP-rtTA/Tet-op-K-Ras4bG12D bitransgenic mice) in the presence and absence of pulmonary surfactant. Intratracheally administered therapeutic adenovirus with pulmonary surfactant spread to airways, as well as to the alveolar region of the lung, and caused a reduction of lung tumors developed. The therapeutic adenovirus without pulmonary surfactant spread only to airways and was ten-fold less effective in tumor reduction. Here, we demonstrate that pulmonary surfactant is an efficient tool to intratracheally deliver a therapeutic virus to treat KRAS mutation-positive lung cancer in vivo.

Cancer/testis (CT) antigens are expressed in normal germ line tissues and various cancers. They are considered promising target molecules for immunotherapy for patients with various cancers. To identify CT antigens, we performed serological identification of antigens by recombinant expression cloning. The humoral immune response of cancer patients against a newly defined antigen was analyzed. A testicular cDNA library was immunoscreened with serum obtained from a gastric adenocarcinoma patient whose primary cancer had regressed once and most liver metastases had disappeared transiently. We isolated 55 positive cDNA clones comprising 23 different genes. They included 4 genes with testis-specific expression profiles in the Unigene database, including coiled-coil domain containing 62 (CCDC62). RT-PCR analysis showed that the expression of 2 splice variants of CCDC62 was restricted to the testis in normal adult tissues. In malignant tissues, CCDC62 variant 2 (CCDC62-2) was aberrantly expressed in a variety of cancers, including stomach cancer. A serological survey of 191 cancer patients with a range of different cancers by ELISA revealed antibodies to CCDC62-2 in 13 patients, including stomach cancer. None of the 41 healthy donor serum samples were reactive in the same test. The serum reaction against CCDC62-2 was confirmed by western blot. CCDC62-2 is a CT antigen that is immunogenic in cancer patients., ((c) 2008 Wiley-Liss, Inc.)

We have previously shown that the RLakt antigen was predominantly recognized by CD8 cytotoxic T lymphocytes (CTL) in RL male 1-bearing or -rejected syngeneic BALB/c mice. CD8 CTL were directed to the octamer pRL1a peptide IPGLPLSL of which recognition was H-2L(d)-restricted. In this study, we identified a CD4 T-cell epitope peptide in the tumor rejection antigen RLakt on BALB/c radiation-leukemia RL male 1. Analyses of the recognition of a bulk CD4 T-cell line using several recombinant RLakt proteins suggested the presence of multiple CD4 T-cell epitopes in the molecule. However, cloning from a bulk CD4 T-cell line resulted in only two clones from 200 wells seeded at three cells per well, and those two CD4 T-cell clones recognized the same epitope peptide in RLakt. The epitope peptide was 14-mer p12-25, AYREETLSIIPGLP, and its recognition was H-2IA(d)-restricted. This sequence overlapped with the CD8 T-cell epitope pRL1a in its N-terminal 5 amino acid residues. The relationship of the epitope to the pRL1a peptide predominantly recognized by CD8 CTL suggests that the 14-mer epitope is predominantly recognized by CD4 T-cells.

OY-TES-1 is a novel target that belongs to the family of 'cancer/testis' (CT) antigens. Our goal was to examine the expression and immunogenicity of OY-TES-1 in epithelial ovarian cancer (EOC) to determine its potential as a target for vaccine therapy. OY-TES-1 expression was determined by one-step reverse transcriptase PCR on 100 EOC samples, 5 EOC cell lines, and a panel of normal tissues. Immunohistochemistry (IHC) was performed on the same panel of EOC tissues. Sera from a sub-group of patients were tested for OY-TES-1 antibody by ELISA. Thymus and leukocytes were weakly positive for OY-TES-1 while the remaining 5 normal tissues were negative. Expression of OY-TES-1 by either RT-PCR and/or IHC was demonstrable in 69/100 (69%) tumors. Humoral immunity to OY-TES-1 was demonstrated in 1/10 (10%) serum samples from patients whose tumors expressed the antigen. The median follow-up of the patient population was 34 months. There was no correlation between antigen expression and stage, grade, histology and survival. OY-TES-1 is expressed in 69% of patients with EOC, is absent from normal ovarian tissue, and a proportion of patients show evidence of a specific humoral immune response. These findings make OY-TES-1 an attractive target for antigen-specific immunotherapy in EOC.

For regulatory factor X4 (RFX4), two alternatively spliced variants, RFX4-A and -B, were reported in the testis. In this study, we identified transcript variants RFX4-C, -D, -E, and -F, and demonstrated by reverse transcription-polymerase chain reaction (RT-PCR) that RFX4-A, -B and -C mRNAs were expressed only in the testis, and RFX4-D mRNA was expressed only in normal brain tissues. In tumors, RFX4-E and -F in addition to RFX4-D mRNA were expressed in gliomas by rapid amplification of cDNA ends and RT-PCR analyses. Expression of RFX4 mRNA was not observed in other tumors, such as lung, esophageal, stomach, colon or liver cancers. Quantitative real-time RT-PCR using common primer pairs detecting all of the variant transcripts showed high expression in normal testis, low expression in the brain (1% compared to the expression in testis), and overexpression in 17 of 61 gliomas (28%). Western blot analysis using DC28 monoclonal antibody (mAb) produced against recombinant RFX4-D C-terminus protein showed expression of RFX4-A and -C proteins, but not RFX4-B protein, in the testis, and expression of RFX4-D protein in the brain. Moreover, expression of RFX4-E and -F proteins, but not RFX4-D protein, was observed in gliomas. Immunohistochemistry analysis using DC28 mAb showed positive staining in the nuclei of spermatocytes in the testis and glioma cells. Antibody against RFX4 was detected in the sera of 3 of 58 (5%) glioma patients by enzyme-linked immunosorbent assay, suggesting the immunogenicity of RFX4-E and -F proteins in glioma patients.

Purpose: XAGE-1 was originally identified by the search for PAGE/GAGE-related genes using expressed sequence tag database and was shown to exhibit characteristics of cancer/testis-like antigens. Four transcript variants XAGE-1a, XAGE-1b, XAGE-1c, and XAGE-1d have been identified thus far. We recently identified XAGE-1b as a dominant antigen recognized by sera from lung adenocarcinoma patients. We here investigated the mRNA expression of four XAGE-1 variants and XAGE-1 protein expression in non-small cell lung cancer (NSCLC). Humoral immune response to XAGE-1b was also evaluated in patients., Experimental Design: Forty-nine NSCLC specimens were analyzed for the expression of four XAGE-1 transcript variants by conventional 30-cycle and real-time reverse transcription-PCR and XAGE-1 protein expression by immunohistochemistry. Sera from 74 patients were analyzed for XAGE-1b antibody production by ELISA and Western blot., Results: XAGE-1b and XAGE-1d mRNA were detected in 15 and 6 of 49 lung cancer specimens, respectively. No XAGE-1a or XAGE-1c mRNA expression was observed. XAGE-1b mRNA expression was observed in 14 of 31 (45%) adenocarcinoma and 1 of 18 (6%) lung cancer with other histologic types. Immunohistochemical analysis using a XAGE-1 monoclonal antibody showed that 14 of 15 XAGE-1b mRNA-positive and 3 of 34 XAGE-1b mRNA-negative specimens expressed XAGE-1 protein. Seropositivity was observed in 5 of 56 patients with adenocarcinoma, whereas none of 18 patients with other histologic types produced XAGE-1b antibody., Conclusion: XAGE-1b is highly and strongly expressed in lung adenocarcinoma and immunogenic in patients, suggesting that XAGE-1b is a promising antigen for immunotherapy against lung adenocarcinoma.

We investigated NY-ESO-1 and LAGE-1a mRNA expression in normal tissues and various types of cancer by quantitative real-time RT-PCR. In addition to their high expression in the testis, we observed a low expression of NY-ESO-1 mRNA in the placenta, pancreas and liver, and no expression in 12 other normal tissues. We also observed a low expression of LAGE-1a mRNA in the placenta and ovary, and marginal expression in 13 other normal tissues. In contrast to the previous finding that NY-ESO-1 and LAGE-1a mRNAs were mostly co-expressed in solid tumors, we found an independent expression of NY-ESO-1 and LAGE-1a mRNAs. NY-ESO-1 mRNA expression was mostly associated with LAGE-1a mRNA expression in esophageal and liver cancers, but not in prostate cancer. Immunohistochemistry (IHC) using NY-ESO-1-specific ES121 mAb showed that NY-ESO-1 protein was detected in 6 of 9 and 3 of 10 NY-ESO-1 mRNA-positive specimens from esophageal and liver cancers, respectively. NY-ESO-1 protein expression was correlated with the copy numbers of NY-ESO-1 mRNA. IHC was also performed using ES121 mAb and B9.8 mAb recognizing both NY-ESO-1 and LAGE-1a in 4 esophageal and 6 liver cancer specimens preferentially expressing LAGE-1a mRNA. B9.8-specific staining was observed weakly and focally in one liver cancer specimen expressing >10(5) copies of LAGE-1a mRNA.

The PHT1 promoter::GUS fusion gene was constructed and introduced into Arabidopsis and rice by Agrobacterium-mediated transformation. Strong beta-glucuronidase (GUS) activity was detected in roots and showed phosphate starvation induction both in Arabidopsis and rice. In contrast, GUS activity in aerial tissues such as those of the leaf and stem was low. In situ GUS staining of root tissue indicated that PHT1 was expressed in root hairs and the outer layer of the main roots, but not in root tips. The PHT1 promoter has a desirable character for biotechnological transgene expression in monocot rice plants.

OY-TES-1 was identified as a human homologue of the mouse, guinea pig, and pig proacrosin binding protein sp32 precursor. Differential expression levels of OY-TES-1 mRNA between testis and other normal tissues, and its expression in cancers indicated that OY-TES-1 would be classified as a cancer/testis antigen and considered to be a candidate of target antigen for cancer immunotherapy. In this study, we showed identification of HLA-A24-binding OY-TES-1 peptide, TES(401-409) (KTPFVSPLL) recognized by CD8 T-cells. Purified CD8 T-cells from healthy donors stimulated in vitro with the peptide-pulsed autologous DC and PBMC produced IFNgamma in response to the peptide-pulsed PBMC and showed cytotoxicity against the peptide-pulsed autologous EBV-B specifically. Furthermore, cytotoxicity was also observed against an OY-TES-1 mRNA-expressing tumor line, LK79. The retention time of the fraction in HPLC of the acid eluate from LK79 cells that showed positive sensitization against autologous EBV-B cells in recognition by CD8 CTL was the same as that of the fraction of the TES(401-409) peptide itself, suggesting that the TES(401-409) was a naturally processed peptide on LK79.

Purpose: Although NY-ESO-1 was isolated from an esophageal carcinoma patient, its expression in this type of cancer and its immunogenicity in esophageal cancer patients have not yet been fully elucidated. We report here the frequency of NY-ESO-1 mRNA and protein expression in esophageal cancer and the presence of NY-ESO-1-specific immune response in patients., Experimental Design: One hundred twenty three esophageal squamous cell carcinoma specimens were analyzed for the expression of NY-ESO-1 mRNA by conventional and real-time reverse transcription-PCR and the expression of protein by immunohistochemistry and Western blot. Sera and peripheral blood lymphocytes from 51 patients were analyzed for the NY-ESO-1 antibody production by enzyme-linked immunosorbent assay and NY-ESO-1 T cell response by enzyme-linked immunospot assay. Survival analyses were also performed., Results: NY-ESO-1 mRNA was expressed in 41 of 123 (33%) esophageal squamous cell carcinoma specimens, and its expression was found at higher frequency in well-differentiated and moderately differentiated type of cancer. No mRNA copy was detected in any of the adjacent normal tissues. Twenty-one of 24 (87.5%) NY-ESO-1 mRNA-positive tumors were stained positively by immunohistochemistry. Correlation between the level of NY-ESO-1 mRNA expression and the degree of immunohistochemistry positivity was observed. Antibody production was observed in 2 patients with tumors that showed protein expression. Furthermore, a CD8 T-cell response against NY-ESO-1 was observed in 1 of the 2 seropositive patients., Conclusions: The high expression frequency of NY-ESO-1 mRNA and protein indicates NY-ESO-1 as a feasible vaccine target in esophageal cancer.

NY-ESO-1 is a cancer/testis antigen expressed in normal adult tissues solely in the testicular germ cells of normal adults and in various cancers. It induces specific humoral and cellular immunity in patients with NY-ESO-1-expressing cancer. The aim of this study was to determine the frequency of NY-ESO-1 mRNA and protein expression in malignant and benign breast tumors. NY-ESO-1 mRNA expression was detected by conventional reverse transcription-PCR and real-time PCR, and that of the protein expression by immunohistochemistry and Western blot analysis. Expression of NY-ESO-1 mRNA was detected in 37 of 88 (42%) cancer specimens, whereas that of the NY-ESO-1 protein was detected only in 1 mRNA-positive specimen. In the latter case, expression level of NY-ESO-1 mRNA relative to that in the testis was relatively high (75% of testicular expression) and to the other among breast cancer specimens. In benign breast lesions, 21 of 31 (68%) specimens expressed low levels of NY-ESO-1 mRNA. In 1 case of fibroadenoma, NY-ESO-1 mRNA was 8% of the testicular level, and protein was detected by Western blot analysis. Only 1 breast cancer patient had detectable antibody at time of surgery, which disappeared within 2 years. Tumor specimen from this patient was both NY-ESO-1 mRNA and protein positive, and NY-ESO-1-specific CD8 T cells were detected in this patient by IFN-gamma enzyme-linked immunospot assay using NY-ESO-1 recombinant adeno and vaccinia virus. A higher rate of NY-ESO-1 expression was noted in breast cancer with high histological grade and negative hormone receptor status, suggesting NY-ESO-1 as a potential tumor antigen for immunotherapy in patients with breast cancer and poor prognosis.

By serologic identification of antigens by recombinant expression cloning (SEREX) analysis using an autologous lung adenocarcinoma cell line, OU-LU-6, as a cDNA library source, we demonstrated that XAGE-1 was the dominant antigen recognized by serum from a patient. By immunoscreening, we obtained 38 positive cDNA clones consisting of 16 genes designated as OY-LC-1 to -OY-LC-16. OY-LC-1, represented by 18 clones, was identical to XAGE-1. OY-LC-2 to -16, represented by either a single or 2 clones, were identical to known genes shown to be ubiquitously expressed in various normal tissues. RT-PCR analysis showed that of 4 XAGE-1 transcripts-XAGE-1a, b, c and d-XAGE-1b was expressed in OU-LU-6 dominantly. Furthermore, XAGE-1b mRNA was expressed in 4 of 10 lung cancer tissues, whereas no expression was observed in normal tissues. Of 4 XAGE-1b mRNA positive cancer tissues, 3 were adenocarcinoma and one was poorly differentiated squamous cell carcinoma. Of 32 sera from lung cancer patients, 8 sera were reactive with the XAGE-1b product. Those 8 sera were from patients with adenocarcinoma. These findings indicated strong immunogenicity of XAGE-1b in lung adenocarcinoma and suggested its potential use as a target for vaccine-based immunotherapies., (Copyright 2003 Wiley-Liss, Inc.)

A DNA vaccine for inducing a tumor immune response was investigated using a well-characterized murine model tumor antigen. We demonstrated that in vivo electroporation augmented the induction of IFNgamma enzyme-linked immunospot (ELISPOT) and cytotoxic T lymphocyte (CTL) generation against pRL1a peptide in BALB/c spleen cells upon immunization with RLakt plasmid. Immunization without in vivo electroporation resulted in only a marginal induction of IFNgamma ELISPOT and CTL generation. Furthermore, co-injection of GM-CSF and RLakt plasmids significantly enhanced the induction of IFNgamma ELISPOT and CTL generation compared to the injection of RLakt plasmid alone. Inhibition of RL male 1 tumor growth was observed by injecting BALB/c mice with GM-CSF and RLakt plasmids using in vivo electroporation, although no effect was observed against an established tumor using the same treatment. No growth inhibition was observed without in vivo electroporation. Immunization with either RLakt plasmid alone, or GM-CSF and pCIneo control plasmids using in vivo electroporation did not inhibit RL male 1 tumor growth.

A-kinase anchoring protein 3 (AKAP3) is a sperm protein and its expression appears to be restricted to the testis in normal adult tissues. We investigated AKAP3 mRNA expression in 20 normal ovaries and 54 ovarian cancers of different histological types, grades and stages by reverse transcription-polymerase chain reaction (RT-PCR). The PCR products were analyzed by conventional agarose gel electrophoresis and capillary electrophoresis on a microtip device to determine the expression semiquantitatively. Little or no expression was observed in the 20 normal ovarian specimens. High AKAP3 mRNA expression was observed in 15 ovarian cancer specimens (28 %). The expression was correlated with the histological grade and clinical stage. AKAP3 mRNA was observed at a significantly higher frequency in poorly differentiated (p = 0.009) and advanced stage (III and IV, p = 0.014) tumors. No correlation was found between AKAP3 mRNA expression and other variables. In Cox multivariate analysis, AKAP3 mRNA expression was found to be a significant predictor of both overall and progression-free survival in patients with poorly differentiated tumors., (Copyright 2004 Wiley-Liss, Inc.)

We showed IgG immune response against endothelial antigens in sera obtained from convalescent Kawasaki disease (KD) patients after recovery from the disease during the follow-up period using serological analysis of recombinant cDNA expression library (SEREX) methodology. We identified 46 antigens represented by 69 clones by immunoscreening of a cDNA expression library from tumor necrosis factor-alpha (TNFalpha) treated human umbilical vein endothelial cells (HUVEC) with sera from 4 KD patients. They included ubiquitin pathway proteins, transcriptional factors, signal transduction molecules, metabolic enzymes, cytoskeletal proteins, an adhesion molecule, and a cell cycle protein. By serological survey using phage plaque assay, sera from 5 non-KD patients were rarely reactive with the antigens. Among the antigens, tropomyosin was most frequently isolated (18 of 69 clones). Seventeen of the 18 clones were identified using KD3 serum. The second most frequently isolated antigen was also a cytoskeletal protein, called Tplastin (3 of 69 clones).

The TSGA10 gene was originally isolated in normal testis by differential mRNA display. TSGA10 is located on chromosome 2q11.2 and consists of 19 exons extending over 3 kb. TSGA10 mRNA expression was investigated in normal and malignant tissues using quantitative real-time RT-PCR. It was predominantly expressed in the testis in adult normal tissues. In malignant tissues, TSGA10 was over-expressed in 4 of 20 hepatocellular carcinomas (HCC), 1 of 20 colon cancers, 7 of 20 ovarian cancers, 3 of 20 prostate cancers, 1 of 21 malignant melanomas, and 8 of 21 bladder cancers. Serological analysis revealed that 3 out of 346 patients with various types of cancer possessed antibody against recombinant TSGA10 protein. They included 2 patients with hepatocellular carcinoma and a patient with malignant melanoma.

NY-ESO-1 mRNA expression was investigated in advanced prostate cancer by conventional and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). NY-ESO-1 mRNA was detected in 20 of 53 (38%) tumor specimens. Four of 15 (27%) stage C, 1 of 3 stage D1 (33%) and 15 of 35 (43%) stage D2 prostate cancers were positive. The presence of NY-ESO-1 antibodies was evaluated in sera from a panel of 218 patients with prostate cancer, including the 53 patients whose tumors were examined for NY-ESO-1 mRNA expression. NY-ESO-1 antibodies were detected in 1 of 30 (3.3%) stage D1 and 9 of 110 (8.2%) stage D2 patients, whereas none of the 78 patients with localized prostate cancer (stages A, B and C) had detectable NY-ESO-1 antibodies. Of the 53 patients whose tumors were examined for NY-ESO-1 mRNA expression, 2 of 20 patients with NY-ESO-1 mRNA-positive tumors had NY-ESO-1 antibodies. No antibody was found in the sera of 32 patients with NY-ESO-1 mRNA-negative tumors, with the exception of one patient with regional lymph node metastasis (stage D1). CD8 T cell responses specific to NY-ESO-1 were detected in two of three patients with NY-ESO-1 antibodies.

Background: To identify antigens specifically recognized by the immune surveillance system in patients with hepatocellular carcinoma (HCC), the authors examined two complementary DNA (cDNA) libraries of moderately differentiated HCC by serologic analysis of recombinant cDNA expression libraries (SEREX)., Methods: The libraries were screened with autologous patients' sera, and sequences of the reacted clones were determined. To study the immunoreactivity of the antigens, sera from 20 patients with HCC, from 20 healthy volunteers, and from 16 patients with chronic viral hepatitis were examined., Results: Twenty-seven antigens were identified. They included SART1, p57Kip2, ROCK-1, gamma-catenin, and heat shock proteins, which are classified as tumor-associated genes. Three of 27 antigens-Tat-binding protein-1 (TBP-1), beta4 integrin-binding protein (p27[BBP]), and ribosomal protein L30 (rpL30)-were reacted predominantly with sera from patients with HCC (55% of patients, 45% of patients, and 20% of patients, respectively). Patients in the control group had no antibodies against these three antigens. Seventy percent of patients with HCC had the antibody against at least one of these antigens., Conclusions: Disease specific humoral immune response against TBP-1, p27(BBP), and rpL30 was induced in patients with HCC, and the antibodies against these antigens also may be used as tumor markers., (Copyright 2003 American Cancer Society.DOI 10.1002/cncr.11374)

Using the recently developed ELISPOT cloning methodology, we obtained cDNA clone S35 coding for the Ag epitope recognized by a murine sarcoma Meth A-specific CTL clone AT-1. Analysis of truncated S35 constructs and overlapping peptides revealed that the peptide epitope was LGAEAIFRL. AT-1 CTL lysed peptide-pulsed CMS8 cells at a nanomolar concentration, and the peptide strongly stimulated IFN-gamma production in AT-1 CTL. Sequence homology indicated that the S35 was derived from a mouse homologue of human retinoic acid-regulated nuclear matrix-associated protein (ramp). The ramp gene consisted of 15 exons. The majority of the ramp mRNA was the transcript normally spliced between exons 14 and 15, but a minor population of mRNA with an extended exon 14 was also present in Meth A cells. The epitope was derived from the newly created open reading frame, which resulted from extension of exon 14 after splicing of the adjacent intronic sequence.

We investigated the generation of CD8 cytotoxic T-lymphocytes (CTL) that recognized a dominant pRL1a peptide bound to H-2L(d) molecule on RL male 1 leukemia in spleen cells from RL male 1-bearing syngeneic BALB/c, semiallogeneic CB6F1 and allogeneic B6 mice by repetitive in vitro stimulation with RL male 1 tumor. CD8 T cells in cultures were also analyzed by H-2L(d)/pRL1a tetramer staining. We showed that pRL1a-specific CTL were more efficiently generated in spleen cells from RL male 1-bearing high responder CB6F1 mice than in low responder BALB/c mice, and this correlated well with the occurrence of H-2L(d)/pRL1a tetramer binding CD8 T cells. Furthermore, we showed that in spleen cells from RL male 1-bearing allogeneic B6 mice, H-2L(d)/pRL1a complex specific CD8 T cells were present at a significant frequency. H-2L(d)/pRL1a recognizing B6 CTL but not BALB/c or CB6F1 CTL gradually lost CD8 expression on their surface by multiplication of in vitro stimulation.

We showed that pRL1a multiple antigen peptide (MAP)-sensitized dendritic cell (DC) and P815 cell lysis by pRL1a-specific B-24 CTL was blocked by incubating target cells at 4 degrees C during sensitization. The finding suggested that pRL1a MAP was mostly internalized in DC and P815 cells and produced pRL1a peptide epitopes for presentation with H-2L(d). Furthermore, we showed that sensitization with pRL1a MAP was inhibited by the addition of chloroquine, cycloheximide, and brefeldin A to the culture, but not by the addition of inhibitors for lysosomal proteases or proteasome. Inhibition of sensitization by the addition of chloroquine to the culture suggested the requirement of acidification of the endosomal compartment for pRL1a MAP processing. Inhibition of sensitization by the addition of cycloheximide and brefeldin A to the culture indicated the requirement of newly generated MHC class I antigen molecules and the involvement of transport of the peptide MHC class I complex from the endoplasmic reticulum to the Golgi. The findings suggested that pRL1a MAP in the endosomal compartment leaked to the cytosol, and degraded, and the pRL1a peptide produced was presented by the MHC class I pathway.