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1.  A phase I study of combination vaccine treatment of five therapeutic epitope-peptides for metastatic colorectal cancer; safety, immunological response, and clinical outcome 
Background
To evaluate the safety of combination vaccine treatment of multiple peptides, phase I clinical trial was conducted for patients with advanced colorectal cancer using five novel HLA-A*2402-restricted peptides, three peptides derived from oncoantigens, ring finger protein 43 (RNF43), 34 kDa-translocase of the outer mitochondrial membrane (TOMM34), and insulin-like growth factor–II mRNA binding protein 3 (KOC1), and the remaining two from angiogenesis factors, vascular endothelial growth factor receptor 1 (VEGFR1) and VEGFR2.
Methods
Eighteen HLA- A*2402-positive colorectal cancer patients who had failed to standard therapy were enrolled in this study. 0.5 mg, 1.0 mg or 3.0 mg each of the peptides was mixed with incomplete Freund’s adjuvant and then subcutaneously injected at five separated sites once a week. We also examined possible effect of a single site injection of “the cocktail of 5 peptides” on the immunological responses. ELISPOT assay was performed before and after vaccinations in the schedule of every 4 weeks.
Results
The vaccine treatment using multiple peptides was well tolerated without any severe treatment-associated systemic adverse events. Dose-dependent induction of peptide-specific cytotoxic T lymphocytes was observed. The single injection of “peptides cocktail” did not diminish the immunological responses. Regarding the clinical outcome, one patient achieved complete response and 6 patients revealed stable disease for 4 to 7 months. The median overall survival time (MST) was 13.5 months. Patients, in which we detected induction of cytotoxic T lymphocytes specific to 3 or more peptides, revealed significantly better prognosis (MST; 27.8 months) than those with poorer immune responses (MST; 3.7 months) (p = 0.032).
Conclusion
Our cancer vaccine treatment using multiple peptides is a promising approach for advanced colorectal cancer with the minimum risk of systemic adverse reactions.
Clinical trial registration
UMIN-CTR number UMIN000004948.
doi:10.1186/1479-5876-12-63
PMCID: PMC4007571  PMID: 24612787
Peptide vaccine; Peptide cocktail; Colorectal cancer; Phase I study
2.  Phase I clinical trial of multiple-peptide vaccination for patients with advanced biliary tract cancer 
Background
The prognosis of patients with advanced biliary tract cancer (BTC) is extremely poor and only a few standard treatments are available for this condition. We performed a phase I trial to investigate the safety, immune response and anti-tumor effect of vaccination with three peptides derived from cancer-testis antigens.
Methods
This study was conducted as a phase I trial. Nine patients with advanced BTC who had unresectable tumors and were refractory to standard chemotherapy were enrolled. Three HLA-A*2402 restricted epitope peptides-cell division cycle associated 1 (CDCA1), cadherin 3 (CDH3) and kinesin family member 20A (KIF20A)-were administered subcutaneously, and the adverse events and immune response were assessed. The clinical effects observed were the tumor response, progression-free survival (PFS) and overall survival (OS).
Results
The three-peptide vaccination was well-tolerated up to a dose of 3 mg per peptide (9 mg total). No grade 3 or 4 adverse events were observed after vaccination. Peptide-specific T cell immune responses were observed in all patients and stable disease was observed in 5 of 9 patients. The median PFS and OS were 3.4 and 9.7 months. The Grade 2 injection site reaction and continuous vaccination after PD judgment appeared to be prognostic of OS.
Conclusions
Multiple-peptide vaccination was well tolerated and induced peptide-specific T-cell responses.
Trial registration
This study was registered with the University Hospital Medical Information Network Clinical Trials Registry (UMIN-CTR000003229).
doi:10.1186/1479-5876-12-61
PMCID: PMC4015445  PMID: 24606884
Cancer vaccine; Peptide vaccine; Immunotherapy; Biliary tract cancer
3.  Identification of an HLA-A2-Restricted Epitope Peptide Derived from Hypoxia-Inducible Protein 2 (HIG2) 
PLoS ONE  2014;9(1):e85267.
We herein report the identification of an HLA-A2 supertype-restricted epitope peptide derived from hypoxia-inducible protein 2 (HIG2), which is known to be a diagnostic marker and a potential therapeutic target for renal cell carcinoma. Among several candidate peptides predicted by the HLA-binding prediction algorithm, HIG2-9-4 peptide (VLNLYLLGV) was able to effectively induce peptide-specific cytotoxic T lymphocytes (CTLs). The established HIG2-9-4 peptide-specific CTL clone produced interferon-γ (IFN-γ) in response to HIG2-9-4 peptide-pulsed HLA-A*02:01-positive cells, as well as to cells in which HLA-A*02:01 and HIG2 were exogenously introduced. Moreover, the HIG2-9-4 peptide-specific CTL clone exerted cytotoxic activity against HIG2-expressing HLA-A*02:01-positive renal cancer cells, thus suggesting that the HIG2-9-4 peptide is naturally presented on HLA-A*02:01 of HIG-2-expressing cancer cells and is recognized by CTLs. Furthermore, we found that the HIG2-9-4 peptide could also induce CTLs under HLA-A*02:06 restriction. Taken together, these findings indicate that the HIG2-9-4 peptide is a novel HLA-A2 supertype-restricted epitope peptide that could be useful for peptide-based immunotherapy against cancer cells with HIG2 expression.
doi:10.1371/journal.pone.0085267
PMCID: PMC3885709  PMID: 24416375
4.  Immunological responses to a multi-peptide vaccine targeting cancer-testis antigens and VEGFRs in advanced pancreatic cancer patients 
Oncoimmunology  2013;2(11):e27010.
The prognosis of patients with advanced pancreatic cancer is extremely poor and there are only a few standard treatments. Here, we report the results of a Phase I clinical trial to investigate the safety, immunostimulatory effects, and antineoplastic activity of a multi-target vaccine composed of four distinct peptides derived from cancer-testis (CT) antigens and vascular endothelial growth factor receptors (VEGFRs). Nine patients with unresectable, advanced pancreatic cancer who were refractory to standard chemotherapy were enrolled. Each patient was vaccinated with HLA-A*2402-restricted peptides derived from the CT antigens kinesin family member 20A (KIF20A) and cell division cycle-associated 1 (CDCA1) as well as from VEGFR1 and VEGFR2 subcutaneously once a week, and disease progression was evaluated up to 6 mo later. Adverse events were assessed using the Common Terminology Criteria for Adverse Events v. 3.0. Immunological responses were monitored by ELISPOT assays and flow cytometry based on peptide-specific dextramers. The clinical outcomes that were measured were tumor response, progression-free survival (PFS) and overall survival (OS). In general, the multi-peptide vaccine was well-tolerated, and no grade 3 or 4 adverse events were observed upon vaccination. Peptide-specific T-cell responses were detected in all 9 patients, and clinical benefits were observed in four of them. Median PFS and OS were 90 and 207 d, respectively. The elicitation of multiple and robust peptide-specific T-cell responses as well as the status of host lymphocytes may be useful prognostic factors among patients with advanced pancreatic cancer treated with peptide-based anticancer vaccines.
doi:10.4161/onci.27010
PMCID: PMC3906430  PMID: 24498547
cancer-testis antigens; clinical trial; immunotherapy; multi-target vaccine; pancreatic cancer; peptide; VEGFR
5.  Local Effects of Regulatory T Cells in MUC1 Transgenic Mice Potentiate Growth of MUC1 Expressing Tumor Cells In Vivo 
PLoS ONE  2012;7(9):e44770.
MUC1 transgenic (MUC1.Tg) mice have widely been used as model recipients of cancer immunotherapy with MUC1. Although MUC1.Tg mice have previously been shown to be immunologically tolerant to MUC1, the involvement of regulatory T (Treg) cells in this phenotype remains unclear. Here, we showed that numbers of Treg cells in MUC1-expressing tumors were greater in MUC1.Tg mice than in control C57BL/6 (B6) mice, and that the growth of tumor cells expressing MUC1, but not that of control cells, in MUC1. Tg mice was faster than in B6 mice. The MUC1.Tg mice appeared to develop MUC1-specific peripheral tolerance, as transferred MUC1-specific T cells were unable to function in MUC1.Tg mice but were functional in control B6 mice. The suppressive function of CD4+CD25high cells from MUC1.Tg mice was more potent than that of cells from control B6 mice when Treg cell activity against MUC1-specific T cells was compared in vitro. Therefore, the enhanced growth of MUC1-expressing tumor cells in MUC1.Tg mice is likely due to the presence of MUC1-specific Treg cells.
doi:10.1371/journal.pone.0044770
PMCID: PMC3444443  PMID: 23028615
6.  Decrease of Peripheral and Intestinal NKG2A-Positive T Cells in Patients with Ulcerative Colitis 
PLoS ONE  2012;7(9):e44113.
To investigate the role of inhibitory natural killer receptors (iNKRs) in inflammatory bowel disease (IBD), we analyzed the expression of NKG2A, one of the iNKRs, on T cells in a mouse colitis model and human IBD. During the active phase of dextran sulfate sodium (DSS)-induced mouse colitis, the frequency of NKG2A+ T cells was significantly decreased in the peripheral blood, and increased in the intestine, suggesting the mobilization of this T cell subset to the sites of inflammation. Administration of anti-NKG2A antibody increased the number of inflammatory foci in DSS-induced colitis, suggesting the involvement of NKG2A+ T cells in this colitis model. In ulcerative colitis (UC) patients, the frequency of peripheral blood NKG2A+ T cells was significantly decreased, compared with Crohn's disease (CD) patients and healthy controls, regardless of clinical conditions such as treatment modalities and disease activity. Notably, in sharp contrast to the DSS-induced mouse colitis model, the frequency of NKG2A+ cells among intestinal T cells was also decreased in UC patients. These results suggest that inadequate local infiltration of NKG2A+ T cells may be involved in the pathogenesis of UC.
doi:10.1371/journal.pone.0044113
PMCID: PMC3435414  PMID: 22970169
7.  Factors responsible for deep-sowing tolerance in wheat seedlings: varietal differences in cell proliferation and the co-ordinated synchronization of epidermal cell expansion and cortical cell division for the gibberellin-mediated elongation of first internodes 
Annals of Botany  2011;108(3):439-447.
Background and Aims
A wheat cultivar, Triticum aestivum ‘Hong Mang Mai’, shows tolerance to deep-sowing conditions by extreme elongation of the first internode, likely mediated by the gibberellin (GA) response. To understand factors involved in the response of this deep-sowing-tolerant cultivar, cell expansion and division that confer elongation on the first internodes of wheat seedlings were investigated.
Methods
The lengths and numbers of epidermal and cortical cells of the first internodes in three wheat cultivars were measured. These parameters were compared in wheat seedlings treated with gibberellin A3 (GA3) or an inhibitor of GA biosynthesis, uniconazole.
Key Results
The varietal differences in the elongation of the first internodes were due to differences in cell numbers resulting from the different abilities of cell division, but not cell expansion. In seedlings treated with GA3, the first internode of ‘Hong Mang Mai’ was 2-fold longer than the control. The GA-stimulated elongation of the first internodes was attributed to 2-fold increases in the number of cortical cells and length of epidermal cells. The different GA-responses observed in these two tissues were also detected in other cultivars, although the response was much lower than that noted in ‘Hong Mang Mai’. The seedlings treated with uniconazole exhibited reduced numbers of cortical cells and reduced lengths of epidermal cells, with both of these effects being more pronounced in ‘Hong Mang Mai’.
Conclusions
The deep-sowing-tolerant cultivar ‘Hong Mang Mai’ is able to elongate the first internode to a greater degree due to enhanced cell division and a heightened response to GA. In addition, cell expansion in the epidermis and cell division in the cortex are synchronized for the elongation of the first internodes. In response to GA, this well-co-ordinated synchronization yields the rapid elongation of the first internodes in wheat seedlings.
doi:10.1093/aob/mcr173
PMCID: PMC3158689  PMID: 21791455
Cell expansion; cell division; deep-sowing tolerance; first internode elongation; gibberellin (GA); ‘Hong Mang Mai’; Triticum aestivum; wheat
8.  Multicenter, phase II clinical trial of cancer vaccination for advanced esophageal cancer with three peptides derived from novel cancer-testis antigens 
Background
Since a phase I clinical trial using three HLA-A24-binding peptides from TTK protein kinase (TTK), lymphocyte antigen-6 complex locus K (LY6K), and insulin-like growth factor-II mRNA binding protein-3 (IMP3) had been shown to be promising for esophageal squamous cell carcinoma (ESCC), we further performed a multicenter, non-randomized phase II clinical trial.
Patients and methods
Sixty ESCC patients were enrolled to evaluate OS, PFS, immunological response employing ELISPOT and pentamer assays. Each of the three peptides was administered with IFA weekly. All patients received the vaccination without knowing an HLA-A type, and the HLA types were key-opened at the analysis point. Hence, the endpoints were set to evaluate differences between HLA-A*2402-positive (24(+)) and -negative (24(−)) groups.
Results
The OS in the 24 (+) group (n = 35) tended to be better than that in the 24(−) group (n = 25) (MST 4.6 vs. 2.6 month, respectively, p = 0.121), although the difference was not statistically significant. However, the PFS in the 24(+) group was significantly better than that in the 24(−) group (p = 0.032). In the 24(+) group, ELISPOT assay indicated that the LY6K-, TTK-, and IMP3-specific CTL responses were observed after the vaccination in 63%, 45%, and 60% of the 24(+) group, respectively. The patients having LY6K-, TTK-, and IMP3-specific CTL responses revealed the better OS than those not having CTL induction, respectively. The patients showing the CTL induction for multiple peptides have better clinical responses.
Conclusions
The immune response induced by the vaccination could make the prognosis better for advanced ESCC patients.
Trial registration
ClinicalTrials.gov, number NCT00995358
doi:10.1186/1479-5876-10-141
PMCID: PMC3403921  PMID: 22776426
Cancer vaccine; Esophageal cancer; Phase II clinical trial; CTL; Peptide vaccine
9.  Identification of HLA-A24-Restricted Novel T Cell Epitope Peptides Derived from P-Cadherin and Kinesin Family Member 20A 
We here identified human leukocyte antigen-(HLA-)A∗2402-restricted epitope peptides from Cadherin 3, type 1, P-cadherin (CDH3) and kinesin family member 20A (KIF20A) that were found to be specifically expressed in cancer cells through genome-wide expression profile analysis. CDH3-10-807 peptide and KIF20A-10-66 peptide successfully induced specific CTL clones, and these selectively responded to COS7 cells expressing both HLA-A∗2402 and respective protein while did not respond to parental cells or COS7 cells expressing either HLA-A∗2402 or respective protein. Furthermore, CTL clones responded to cancer cells that endogenously express HLA-A∗2402 and respective protein, suggesting that CDH3-10-807 peptide and KIF20A-10-66 peptide are naturally presented on HLA-A∗2402 molecule of human cancer cells. Our results demonstrated that CDH3-10-807 peptide and KIF20A-10-66 peptide are novel HLA-A24-restricted tumor-associated antigens and would be applicable for CTL-inducing cancer therapies.
doi:10.1155/2012/848042
PMCID: PMC3388625  PMID: 22778556
10.  Accurate evaluation and verification of varietal ranking for flooding tolerance at the seedling stage in barley (Hordeum vulgare L.) 
Breeding Science  2012;62(1):3-10.
Soil flooding or waterlogging is a major abiotic stress in upland crops. In barley, there have been several reported studies of selection for flooding-tolerant genotypes, but it is difficult to obtain varietal rankings that are consistent among researchers. Our objectives were to establish experimental conditions that could be applied by other research groups and to verify the varietal ranking conducted in an earlier study. We conducted greenhouse experiments on 14 barley varieties. At the 2.5-leaf stage, they were flooded with 0% or 0.1% soluble starch solution (mimicking reducing conditions). At 13 to 15 days after the start of treatment, the degree of leaf injury and the shoot dry weight ratio (treatment:control) were recorded. Reliable and highly repeatable results were obtained for the criterion of leaf injury under reducing conditions, whereas shoot dry weight ratio was unstable. The varieties OUJ820 and OUA301 were highly tolerant, whereas OUA002 and OUJ247 were sensitive; these results matched those of the earlier study. The experimental conditions that we developed here may be useful for selection testing and genetic analysis of flooding tolerance in other laboratories.
doi:10.1270/jsbbs.62.3
PMCID: PMC3405954  PMID: 23136508
barley; flooding; genetic resources; soil reduction; variation; waterlogging
11.  Involvement of Reactive Oxygen Species in Sonodynamically Induced Apoptosis Using a Novel Porphyrin Derivative 
Theranostics  2012;2(9):880-888.
In this study, we investigated the induction of apoptosis by ultrasound in the presence of the novel porphyrin derivative DCPH-P-Na(I). HL-60 cells were exposed to ultrasound for up to 3 min in the presence and absence of DCPH-P-Na(I), and the induction of apoptosis was examined by analyzing cell morphology, DNA fragmentation, and caspase-3 activity. Reactive oxygen species were measured by means of ESR and spin trapping technique. Cells treated with 8 μM DCPH-P-Na(I) and ultrasound clearly showed membrane blebbing and cell shrinkage, whereas significant morphologic changes were not observed in cells exposed to either ultrasound or DCPH-P-Na(I) alone. Also, DNA ladder formation and caspase-3 activation were observed in cells treated with both ultrasound and DCPH-P-Na(I) but not in cells treated with ultrasound or DCPH-P-Na(I) alone. In addition, the combination of DCPH-P-Na(I) and the same acoustical arrangement of ultrasound substantially enhanced nitroxide generation by the cells. Sonodynamically induced apoptosis, caspase-3 activation, and nitroxide generation were significantly suppressed by histidine. These results indicate that the combination of ultrasound and DCPH-P-Na(I) induced apoptosis in HL-60 cells. The significant reduction in sonodynamically induced apoptosis, nitroxide generation, and caspase-3 activation by histidine suggests active species such as singlet oxygen are important in the sonodynamic induction of apoptosis. These experimental results support the possibility of sonodynamic treatment for cancer using the induction of apoptosis.
doi:10.7150/thno.3899
PMCID: PMC3475214  PMID: 23082100
Apoptosis; Sonodynamic therapy; Ultrasound; DCPH-P-Na(I); HL-60 cells, Reactive Oxygen, Caspase-3.
12.  Duplicate polyphenol oxidase genes on barley chromosome 2H and their functional differentiation in the phenol reaction of spikes and grains 
Journal of Experimental Botany  2010;61(14):3983-3993.
Polyphenol oxidases (PPOs) are copper-containing metalloenzymes encoded in the nucleus and transported into the plastids. Reportedly, PPOs cause time-dependent discoloration (browning) of end-products of wheat and barley, which impairs their appearance quality. For this study, two barley PPO homologues were amplified using PCR with a primer pair designed in the copper binding domains of the wheat PPO genes. The full-lengths of the respective PPO genes were cloned using a BAC library, inverse-PCR, and 3′-RACE. Linkage analysis showed that the polymorphisms in PPO1 and PPO2 co-segregated with the phenol reaction phenotype of awns. Subsequent RT-PCR experiments showed that PPO1 was expressed in hulls and awns, and that PPO2 was expressed in the caryopses. Allelic variation of PPO1 and PPO2 was analysed in 51 barley accessions with the negative phenol reaction of awns. In PPO1, amino acid substitutions of five types affecting functionally important motif(s) or C-terminal region(s) were identified in 40 of the 51 accessions tested. In PPO2, only one mutant allele with a precocious stop codon resulting from an 8 bp insertion in the first exon was found in three of the 51 accessions tested. These observations demonstrate that PPO1 is the major determinant controlling the phenol reaction of awns. Comparisons of PPO1 single mutants and the PPO1PPO2 double mutant indicate that PPO2 controls the phenol reaction in the crease on the ventral side of caryopses. An insertion of a hAT-family transposon in the promoter region of PPO2 may be responsible for different expression patterns of the duplicate PPO genes in barley.
doi:10.1093/jxb/erq211
PMCID: PMC2935872  PMID: 20616156
Gene duplication; grasses; Hordeum vulgare; mutant; phenol reaction; PPO
13.  NKG2A inhibits iNKT cell activation in hepatic injury1 
Activation of invariant NKT (iNKT) cells in the liver is generally regarded as the critical step for concanavallin A (Con A)-induced hepatitis, and the role of NK cell receptors for iNKT cell activation is still controversial. Here we show that blockade of the NKG2A-mediated inhibitory signal with antagonistic anti-NKG2A/C/E mAb (20d5) aggravated Con A-induced hepatitis in wild-type, Fas ligand (FasL)-mutant gld, and IL-4-deficient mice even with NK cell- and CD8 T cell-depletion, but not in perforin-, IFN-γ-, or IFN-γ- and perforin-deficient mice. Consistently, 20d5 pre-treatment augmented serum IFN-γ levels and perforin-dependent cytotoxicity of liver mononuclear cells following Con A injection, but not their FasL/Fas-dependent cytotoxicity. However, blockade of NKG2A-mediated signals during the cytotoxicity effector phase did not augment cytotoxic activity. Activated iNKT cells promptly disappeared after Con A injection, while NK1− iNKT cells, that preferentially expressed CD94/NKG2A, predominantly remained in the liver. Pre-treatment with 20d5 appeared to facilitate disappearance of iNKT cell, particularly NK1− iNKT cells. Moreover, Con A-induced and α-galactosylceramide-induced hepatic injury was very severe in CD94/NKG2A-deficient DBA/2J mice compared with CD94/NKG2A-intact DBA/2JJcl mice. Overall, these results indicated that NKG2A-mediated signal negatively regulates iNKT cell activation and hepatic injury.
PMCID: PMC2841747  PMID: 19109156
Rodent; Natural Killer cells; Cell activation
14.  Treating Metastatic Solid Tumors With Bortezomib and a Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand Receptor Agonist Antibody 
Background
Resistance of tumors to cell death signals poses a complex clinical problem. We explored the therapeutic potential and in vivo toxicity of a combination of bortezomib, a proteasome inhibitor, and MD5-1, a tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) receptor (DR5) agonist monoclonal antibody, in mouse carcinomas.
Methods
Mice bearing Renca-FLAG (renal) or 4T1 (mammary) tumors were treated with bortezomib and/or MD5-1 and examined for lung metastases (Renca-FLAG: n = 93; 4T1: n = 40) or monitored for survival (Renca-FLAG: n = 143). Toxicity was assessed by histopathology and hematology. Viability and apoptotic signaling in Renca-FLAG and 4T1 cells treated with bortezomib alone or in combination with TRAIL were analyzed using 3-[4,5-dimethyiazol-2-yl-5]-[3-carboxymethyloxyphenyl]-2-[4-sulfophenyl]-2H tetrazolium assay and by measuring mitochondrial membrane depolarization and caspase-8 and caspase-3 activation. All statistical tests were two-sided.
Results
Bortezomib (20 nM) sensitized Renca-FLAG and 4T1 cells to TRAIL-mediated apoptosis (mean percent decrease in numbers of viable cells, bortezomib + TRAIL vs TRAIL: Renca-FLAG, 95% vs 34%, difference = 61%, 95% confidence interval [CI] = 52% to 69%, P < .001; 4T1, 85% vs 20%, difference = 65%, 95% CI = 62% to 69%, P < .001). Sensitization involved activation of caspase-8 and caspase-3 but not mitochondrial membrane depolarization, suggesting an amplified signaling of the extrinsic cell death pathway. Treatment with bortezomib and MD5-1 reduced lung metastases in mice carrying Renca and 4T1 tumors (mean number of metastases, bortezomib + MD5-1 vs MD5-1: Renca-FLAG, 1 vs 8, difference = 7, 95% CI = 5 to 9, P < .001; 4T1, 1 vs 12, difference = 11, 95% CI = 9 to 12, P < .001) and increased median survival of mice bearing Renca-FLAG tumors (bortezomib + MD5-1 vs bortezomib + control isotype antibody: 22 of 30 [73%] were still alive at day 180 vs median survival of 42 days [95% CI = 41 to 44 days, P < .001]) in the absence of obvious toxicity.
Conclusion
Bortezomib combined with DR5 agonist monoclonal antibody may be a useful treatment for metastatic solid tumors.
doi:10.1093/jnci/djn113
PMCID: PMC2753966  PMID: 18445820
15.  Development of 5006 Full-Length CDNAs in Barley: A Tool for Accessing Cereal Genomics Resources 
A collection of 5006 full-length (FL) cDNA sequences was developed in barley. Fifteen mRNA samples from various organs and treatments were pooled to develop a cDNA library using the CAP trapper method. More than 60% of the clones were confirmed to have complete coding sequences, based on comparison with rice amino acid and UniProt sequences. Blastn homologies (E<1E-5) to rice genes and Arabidopsis genes were 89 and 47%, respectively. Of the 5028 possible amino acid sequences derived from the 5006 FLcDNAs, 4032 (80.2%) were classified into 1678 GreenPhyl multigenic families. There were 555 cDNAs showing low homology to both rice and Arabidopsis. Gene ontology annotation by InterProScan indicated that many of these cDNAs (71%) have no known molecular functions and may be unique to barley. The cDNAs showed high homology to Barley 1 GeneChip oligo probes (81%) and the wheat gene index (84%). The high homology between FLcDNAs (27%) and mapped barley expressed sequence tag enabled assigning linkage map positions to 151–233 FLcDNAs on each of the seven barley chromosomes. These comprehensive barley FLcDNAs provide strong platform to connect pre-existing genomic and genetic resources and accelerate gene identification and genome analysis in barley and related species.
doi:10.1093/dnares/dsn034
PMCID: PMC2671202  PMID: 19150987
full-length cDNA; Hordeum vulgare; mRNA; gene ontology
16.  Immunomodulating Activity of Agaricus brasiliensis KA21 in Mice and in Human Volunteers 
We performed studies on murine models and human volunteers to examine the immunoenhancing effects of the naturally outdoor-cultivated fruit body of Agaricus brasiliensis KA21 (i.e. Agaricus blazei). Antitumor, leukocyte-enhancing, hepatopathy-alleviating and endotoxin shock-alleviating effects were found in mice. In the human study, percentage body fat, percentage visceral fat, blood cholesterol level and blood glucose level were decreased, and natural killer cell activity was increased. Taken together, the results strongly suggest that the A. brasiliensis fruit body is useful as a health-promoting food.
doi:10.1093/ecam/nem016
PMCID: PMC2396466  PMID: 18604247
A. brasiliensis; clinical research; cold water extract; NK activity;  outdoor-cultivated; safety
17.  Induction of Tumor-specific T Cell Immunity by Anti-DR5 Antibody Therapy 
Because tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) preferentially induces apoptosis in tumor cells and plays a critical role in tumor surveillance, its receptor is an attractive target for antibody-mediated tumor therapy. Here we report that a monoclonal antibody (mAb) against the mouse TRAIL receptor, DR5, exhibited potent antitumor effects against TRAIL-sensitive tumor cells in vivo by recruiting Fc receptor–expressing innate immune cells, with no apparent systemic toxicity. Administration of the agonistic anti-DR5 mAb also significantly inhibited experimental and spontaneous tumor metastases. Notably, the anti-DR5 mAb-mediated tumor rejection by innate immune cells efficiently evoked tumor-specific T cell immunity that could also eradicate TRAIL-resistant variants. These results suggested that the antibody-based therapy targeting DR5 is an efficient strategy not only to eliminate TRAIL-sensitive tumor cells, but also to induce tumor-specific T cell memory that affords a long-term protection from tumor recurrence.
doi:10.1084/jem.20031457
PMCID: PMC2211825  PMID: 14769851
apoptosis; macrophage; NK cells; CTL; TRAIL
18.  CAM and NK Cells 
It is believed that tumor development, outgrowth and metastasis are under the surveillance of the immune system. Although both innate and acquired immune systems play roles, innate immunity is the spearhead against tumors. Recent studies have revealed the critical role of natural killer (NK) cells in immune surveillance and that NK cell activity is considerably influenced by various agents, such as environmental factors, stress, foods and drugs. Some of these NK cell stimulants have been used in complementary and alternative medicine (CAM) since ancient times. Therefore, the value of CAM should be re-evaluated from this point of view. In this review, we overview the intimate correlation between NK cell functions and CAM agents, and discuss possible underlying mechanisms mediating this. In particular, neuro-immune crosstalk and receptors for CAM agents are the most important and interesting candidates for such mechanisms.
doi:10.1093/ecam/neh014
PMCID: PMC442116  PMID: 15257322
β-glucan; lectin; NK cell receptor; nerve-immune crosstalk; tumor
19.  Nonredundant Roles for CD1d-restricted Natural Killer T Cells and Conventional CD4+ T Cells in the Induction of Immunoglobulin E Antibodies in Response to Interleukin 18 Treatment of Mice 
The Journal of Experimental Medicine  2003;197(8):997-1005.
Interleukin (IL)-18 synergizes with IL-12 to promote T helper cell (Th)1 responses. Somewhat paradoxically, IL-18 administration alone strongly induces immunoglobulin (Ig)E production and allergic inflammation, indicating a role for IL-18 in the generation of Th2 responses. The ability of IL-18 to induce IgE is dependent on CD4+ T cells, IL-4, and signal transducer and activator of transcription (stat)6. Here, we show that IL-18 fails to induce IgE both in CD1d−/− mice that lack natural killer T (NKT) cells and in class II−/− mice that lack conventional CD4+ T cells. However, class II−/− mice reconstituted with conventional CD4+ T cells show the capacity to produce IgE in response to IL-18. NKT cells express high levels of IL-18 receptor (R)α chain and produce significant amounts of IL-4, IL-9, and IL-13, and induce CD40 ligand expression in response to IL-2 and IL-18 stimulation in vitro. In contrast, conventional CD4+ T cells express low levels of IL-18Rα and poorly respond to IL-2 and IL-18. Nevertheless, conventional CD4+ T cells are essential for B cell IgE responses after the administration of IL-18. These findings indicate that NKT cells might be the major source of IL-4 in response to IL-18 administration and that conventional CD4+ T cells demonstrate their helper function in the presence of NKT cells.
doi:10.1084/jem.20021701
PMCID: PMC2193873  PMID: 12695491
IL-18R; CD4+ NK1.1+ T cells; Th2 cytokines; CD40 ligand; allergy
20.  Activation of Natural Killer T Cells by α-Galactosylceramide Impairs DNA Vaccine-Induced Protective Immunity against Trypanosoma cruzi  
Infection and Immunity  2003;71(3):1234-1241.
Innate immunity as a first defense is indispensable for host survival against infectious agents. We examined the roles of natural killer (NK) T cells in defense against Trypanosoma cruzi infection. The T. cruzi parasitemia and survival of CD1d-deficient mice exhibited no differences compared to wild-type littermates. NK T-cell activation induced by administering α-galactosylceramide (α-GalCer) to T. cruzi-infected mice significantly changed the parasitemia only in the late phase of infection and slightly improved survival when mice were infected intraperitoneally. The combined usage of α-GalCer and benznidazole, a commercially available drug for Chagas' disease, did not enhance the therapeutic efficacy of benznidazole. These results suggest that NK T cells do not play a pivotal role in resistance to T. cruzi infection. In addition, we found that the coadministration of α-GalCer with DNA vaccine impaired the induction of epitope-specific CD8+ T cells and undermined the DNA vaccine-induced protective immunity against T. cruzi. Our results, in contrast to previous reports demonstrating the protective roles of NK T cells against other infectious agents, suggest that these cells might even exhibit adverse effects on vaccine-mediated protective immunity.
doi:10.1128/IAI.71.3.1234-1241.2003
PMCID: PMC148846  PMID: 12595437
21.  Coadministration of an Interleukin-12 Gene and a Trypanosoma cruzi Gene Improves Vaccine Efficacy  
Infection and Immunity  2002;70(9):4833-4840.
We tested the immunogenicity of two Trypanosoma cruzi antigens injected into mice in the form of DNA vaccine. Immunization with DNA encoding dihydroorotate dehydrogenase did not confer protective immunity in all mouse strains tested. Immunization with DNA encoding trans-sialidase surface antigen (TSSA) protected C57BL/6 (H-2b) mice but not BALB/c (H-2d) or C3H/Hej (H-2k) mice against lethal T. cruzi infection. In vivo depletion of CD4+ or CD8+ T cells abolished the protective immunity elicited by TSSA gene in C57BL/6 mice. Enzyme-linked immunospot assay with splenocytes from T. cruzi-infected mice or TSSA gene-vaccinated mice identified an H-2Kb-restricted antigenic peptide, ANYNFTLV. The CD8+-T-cell line specific for this peptide could recognize T. cruzi-infected cells in vitro and could protect naive mice from lethal infection when adoptively transferred. Coadministration of the interleukin-12 (IL-12) gene with the TSSA gene facilitated the induction of ANYNFTLV-specific CD8+ T cells and improved the vaccine efficacy against lethal T. cruzi infection. These results reinforced the utility of immunomodulatory adjuvants such as IL-12 gene for eliciting protective immunity against intracellular parasites by DNA vaccination.
doi:10.1128/IAI.70.9.4833-4840.2002
PMCID: PMC128276  PMID: 12183527
22.  Critical Role for Tumor Necrosis Factor–related Apoptosis-inducing Ligand in Immune Surveillance Against Tumor Development 
Natural killer (NK) cells and interferon (IFN)-γ have been implicated in immune surveillance against tumor development. Here we show that tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) plays a critical role in the NK cell–mediated and IFN-γ–dependent tumor surveillance. Administration of neutralizing monoclonal antibody against TRAIL promoted tumor development in mice subcutaneously inoculated with a chemical carcinogen methylcholanthrene (MCA). This protective effect of TRAIL was at least partly mediated by NK cells and totally dependent on IFN-γ. In the absence of TRAIL, NK cells, or IFN-γ, TRAIL-sensitive sarcomas preferentially emerged in MCA-inoculated mice. Moreover, development of spontaneous tumors in p53+/− mice was also promoted by neutralization of TRAIL. These results indicated a substantial role of TRAIL as an effector molecule that eliminates developing tumors.
doi:10.1084/jem.20011171
PMCID: PMC2193611  PMID: 11805143
NK cells; IFN-γ; methylcholanthrene-induced fibrosarcoma; p53; innate immune response
23.  Tumor Necrosis Factor–Related Apoptosis-Inducing Ligand (Trail) Contributes to Interferon γ–Dependent Natural Killer Cell Protection from Tumor Metastasis 
Tumor necrosis factor–related apoptosis-inducing ligand (TRAIL) is expressed by in vitro activated natural killer (NK) cells, but the relevance of this observation to the biological function of NK cells has been unclear. Herein, we have demonstrated the in vivo induction of mouse TRAIL expression on various tissue NK cells and correlated NK cell activation with TRAIL-mediated antimetastatic function in vivo. Expression of TRAIL was only constitutive on a subset of liver NK cells, and innate NK cell control of Renca carcinoma hepatic metastases in the liver was partially TRAIL dependent. Administration of therapeutic doses of interleukin (IL)-12, a powerful inducer of interferon (IFN)-γ production by NK cells and NKT cells, upregulated TRAIL expression on liver, spleen, and lung NK cells, and IL-12 suppressed metastases in both liver and lung in a TRAIL-dependent fashion. By contrast, α-galactosylceramide (α-GalCer), a powerful inducer of NKT cell IFN-γ and IL-4 secretion, suppressed both liver and lung metastases but only stimulated NK cell TRAIL-mediated function in the liver. TRAIL expression was not detected on NK cells from IFN-γ–deficient mice and TRAIL-mediated antimetastatic effects of IL-12 and α-GalCer were strictly IFN-γ dependent. These results indicated that TRAIL induction on NK cells plays a critical role in IFN-γ–mediated antimetastatic effects of IL-12 and α-GalCer.
PMCID: PMC2193421  PMID: 11257133
rodent; tumor immunity; in vivo animal models; immunotherapy; interleukin 12
24.  Critical Contribution of Ox40 Ligand to T Helper Cell Type 2 Differentiation in Experimental Leishmaniasis 
Infection of inbred mouse strains with Leishmania major is a well characterized model for analysis of T helper (Th)1 and Th2 cell development in vivo. In this study, to address the role of costimulatory molecules CD27, CD30, 4-1BB, and OX40, which belong to the tumor necrosis factor receptor superfamily, in the development of Th1 and Th2 cells in vivo, we administered monoclonal antibody (mAb) against their ligands, CD70, CD30 ligand (L), 4-1BBL, and OX40L, to mice infected with L. major. Whereas anti-CD70, anti-CD30L, and anti–4-1BBL mAb exhibited no effect in either susceptible BALB/c or resistant C57BL/6 mice, the administration of anti-OX40L mAb abrogated progressive disease in BALB/c mice. Flow cytometric analysis indicated that OX40 was expressed on CD4+ T cells and OX40L was expressed on CD11c+ dendritic cells in the popliteal lymph nodes of L. major–infected BALB/c mice. In vitro stimulation of these CD4+ T cells showed that anti-OX40L mAb treatment resulted in substantially reduced production of Th2 cytokines. Moreover, this change in cytokine levels was associated with reduced levels of anti–L. major immunoglobulin (Ig)G1 and serum IgE. These results indicate that anti-OX40L mAb abrogated progressive leishmaniasis in BALB/c mice by suppressing the development of Th2 responses, substantiating a critical role of OX40–OX40L interaction in Th2 development in vivo.
PMCID: PMC2195752  PMID: 10637281
OX40/OX40 ligand; experimental leishmaniasis; Th1/Th2 differentiation; costimulation; TNF/TNF receptor family
25.  The Natural Killer T (NKT) Cell Ligand α-Galactosylceramide Demonstrates Its Immunopotentiating Effect by Inducing Interleukin (IL)-12 Production by Dendritic Cells and IL-12 Receptor Expression on NKT Cells  
The Journal of Experimental Medicine  1999;189(7):1121-1128.
The natural killer T (NKT) cell ligand α-galactosylceramide (α-GalCer) exhibits profound antitumor activities in vivo that resemble interleukin (IL)-12–mediated antitumor activities. Because of these similarities between the activities of α-GalCer and IL-12, we investigated the involvement of IL-12 in the activation of NKT cells by α-GalCer. We first established, using purified subsets of various lymphocyte populations, that α-GalCer selectively activates NKT cells for production of interferon (IFN)-γ. Production of IFN-γ by NKT cells in response to α-GalCer required IL-12 produced by dendritic cells (DCs) and direct contact between NKT cells and DCs through CD40/CD40 ligand interactions. Moreover, α-GalCer strongly induced the expression of IL-12 receptor on NKT cells from wild-type but not CD1−/− or Vα14−/− mice. This effect of α-GalCer required the production of IFN-γ by NKT cells and production of IL-12 by DCs. Finally, we showed that treatment of mice with suboptimal doses of α-GalCer together with suboptimal doses of IL-12 resulted in strongly enhanced natural killing activity and IFN-γ production. Collectively, these findings indicate an important role for DC-produced IL-12 in the activation of NKT cells by α-GalCer and suggest that NKT cells may be able to condition DCs for subsequent immune responses. Our results also suggest a novel approach for immunotherapy of cancer.
PMCID: PMC2193012  PMID: 10190903
natural killer T cells; dendritic cells; α-galactosylceramide; interleukin 12; interleukin 12 receptor

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