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Supplementary Figure Legend from Treatment with Chemotherapy and Dendritic Cells Pulsed with Multiple Wilms' Tumor 1 (WT1)–Specific MHC Class I/II–Restricted Epitopes for Pancreatic Cancer

Adverse events during the first 3 courses of treatment.

Frequency of regulatory T cells or myeloid-derived suppressor cells in patients before and after therapy.

Purpose: We performed a phase I trial to investigate the safety, clinical responses, and Wilms' tumor 1 (WT1)-specific immune responses following treatment with dendritic cells (DC) pulsed with a mixture of three types of WT1 peptides, including both MHC class I and II–restricted epitopes, in combination with chemotherapy.Experimental Design: Ten stage IV patients with pancreatic ductal adenocarcinoma (PDA) and 1 patient with intrahepatic cholangiocarcinoma (ICC) who were HLA-positive for A*02:01, A*02:06, A*24:02, DRB1*04:05, DRB1*08:03, DRB1*15:01, DRB1*15:02, DPB1*05:01, or DPB1*09:01 were enrolled. The patients received one course of gemcitabine followed by biweekly intradermal vaccinations with mature DCs pulsed with MHC class I (DC/WT1-I; 2 PDA and 1 ICC), II (DC/WT1-II; 1 PDA), or I/II–restricted WT1 peptides (DC/WT1-I/II; 7 PDA), and gemcitabine.Results: The combination therapy was well tolerated. WT1-specific IFNγ-producing CD4+ T cells were significantly increased following treatment with DC/WT1-I/II. WT1 peptide-specific delayed-type hypersensitivity (DTH) was detected in 4 of the 7 patients with PDA vaccinated with DC/WT1-I/II and in 0 of the 3 patients with PDA vaccinated with DC/WT1-I or DC/WT1-II. The WT1-specific DTH-positive patients showed significantly improved overall survival (OS) and progression-free survival (PFS) compared with the negative control patients. In particular, all 3 patients with PDA with strong DTH reactions had a median OS of 717 days.Conclusions: The activation of WT1-specific immune responses by DC/WT1-I/II combined with chemotherapy may be associated with disease stability in advanced pancreatic cancer. Clin Cancer Res; 20(16); 4228–39. ©2014 AACR.

Natural astaxanthin is known to be produced by green microalgae, a potent producer of the most powerful antioxidant. To increase the productivity of astaxanthin in microalgae, random mutagenesis has been extensively used to improve the yield of valuable substances. In the presented work, a newly isolated Coelastrum sp. was randomly mutagenized by exposure to ethyl methane sulfonate and further screened using two approaches; an approach for high growth mutant and an approach for high astaxanthin producing mutant with a high-throughput screening method using glufosinate. Among these, mutant G1-C1 that was selected using glufosinate showed the highest of total carotenoids (45.48±1.5 mg/L) and astaxanthin (28.32±2.5 mg/L) production, which was almost 2-fold higher than that of wild type. This study indicates that random mutagenesis via chemical mutation strategy and screening using glufosinate successfully expedited astaxanthin production in a mutated strain of a Coelastrum sp.

Alien, introduced, and non-native species expand their distribution through the development of various transportation networks. In Japan, Ligustrum lucidum Aiton (Oleaceae) was introduced in the mid-1800s for use as roadside trees, but has escaped from the planting area and spread everywhere. This species also invades coastal areas and has adapted to develop its characteristic features under various stress conditions. To clarify the adaptive features of L. lucidum in coastal areas, we conducted morphological and anatomical analyses. We found that the stomatal size of L. lucidum was smaller in the coastal populations than in inland populations, which is similar to the coastal adaptation pattern of L. japonicum Thunb., a closely related native species. Our results suggest that strong selective pressure on coastal area conditions could force their leaves to avoid excessive transpiration, and such an adaptation pattern of L. lucidum could expand its distribution to various coastal areas.

A newly isolated green microalga, Coelastrum sp. has the capability to produce and accumulate astaxanthin under various stress conditions. At present, a mutant G1-C1 of Coelastrum sp. obtained through chemical mutagenesis using ethyl methane sulfonate displayed an improvement in astaxanthin accumulation, which was 2-fold higher than that of the wild-type. However, lack of genomic information limits the understanding of the molecular mechanism that leads to a high level of astaxanthin in the mutant G1-C1. In this study, transcriptome sequencing was performed to compare the transcriptome of astaxanthin hyper-producing mutant G1-C1 and wild-type of Coelastrum sp. with respect to astaxanthin biosynthesis. This is to clarify why the mutant produced higher astaxanthin yield compared to the wild-type strain. Based on the transcriptomic analysis, the differentially expressed genes involved in astaxanthin biosynthesis were significantly upregulated in the mutant G1-C1 of Coelastrum sp. Genes coding phytoene synthase, phytoene desaturase, ζ-carotene desaturase, and lycopene β-cyclase involved in β-carotene biosynthesis in the mutant cells were upregulated by 10-, 9.2-, 8.4-, and 8.7-fold, respectively. Genes coding beta-carotene ketolase and beta-carotene 3-hydroxylase involved in converting β-carotene into astaxanthin were upregulated by 7.8- and 8.0-fold, respectively. In contrast, the lycopene ε-cyclase gene was downregulated by 9.7-fold in mutant G1-C1. Together, these results contribute to higher astaxanthin accumulation in mutant G1-C1. Overall, the data in this study provided molecular insight for a better understanding of the differences in astaxanthin biosynthesis between the wild-type and mutant G1-C1 strains.

Highlights • A newly isolated Coelastrum sp. improved the yield of astaxanthin by chemical mutagenesis. • Mutagenesis using chemical mutagen of EMS attempted to increase the microalgae biomass and carotenoid production in Coelastrum sp. • High-throughput screening method using glufosinate successfully expedited astaxanthin production in a mutated strain of a Coelastrum sp. • The selected mutant using glufosinate exhibited an increase of astaxanthin content with ∼2-fold higher compared to the wild type., Natural astaxanthin is known to be produced by green microalgae, a potent producer of the most powerful antioxidant. To increase the productivity of astaxanthin in microalgae, random mutagenesis has been extensively used to improve the yield of valuable substances. In the presented work, a newly isolated Coelastrum sp. was randomly mutagenized by exposure to ethyl methane sulfonate and further screened using two approaches; an approach for high growth mutant and an approach for high astaxanthin producing mutant with a high-throughput screening method using glufosinate. Among these, mutant G1-C1 that was selected using glufosinate showed the highest of total carotenoids (45.48±1.5 mg/L) and astaxanthin (28.32±2.5 mg/L) production, which was almost 2-fold higher than that of wild type. This study indicates that random mutagenesis via chemical mutation strategy and screening using glufosinate successfully expedited astaxanthin production in a mutated strain of a Coelastrum sp.

The gut-associated lymphoid tissue (GALT) represents a major reservoir of HIV in infected individuals. Vaccines can induce strong systemic immune responses but these have less impact on CD4 T cells activity and numbers in GALT. In this study, we vaccinated mice with an adenovirus vector that expressed the envelope gene from HIV and observed immune responses in the peripheral blood, spleen, liver, mesenteric lymph nodes, and Peyer's patches. We found that (1) the number of HIV-specific CD8 T cells was dramatically lower in GALT than in other tissues; (2) the programmed cell death protein-1 (PD-1) was expressed at high levels in HIV-specific CD8 T cells including memory T cells in GALT; and (3) high levels of HIV-specific CD8 T cell apoptosis were occurring in GALT. These results suggest that contributing to GALT becoming an HIV reservoir during infection is a combination of exhaustion and/or dysfunction of HIV-specific CTLs at that site. These results emphasize the importance of developing of an effective mucosal vaccine against HIV.

Stimulation of butyrophilin 3 molecules by specific agonistic mAbs results in down-regulation of proliferation and cytokine secretion in CD4 and CD8 T lymphocyte subsets. The BTN molecule consists of three subfamilies, BTN1, BTN2. and BTN3, and possesses interesting properties for biological regulation. Although the biological significance of BTN1 and BTN2 has been progressively clarified, the receptor function of BTN3 remains to be elucidated as a result of the absence of appropriate agonists. To clarify the participation of BTN3 in immune regulation, BTN3-specific mAb, referred to as 34-7 and 232-5, were generated from BTN3 gene-immunized mice. The 232-5 mAb, specific to the extracellular domain of the BTN3 molecule, stained almost all populations of human PBMCs, including T, NK, NKT, and B cells. Notably, treatment with the 232-5 mAb resulted in phosphorylation of BTN3A3 molecules, leading to attenuated proliferation and cytokine secretion by CD4+ and CD8+ T cells in a CD4+ CD25+ Treg cell-independent manner, demonstrating the agonistic property of the 232-5 mAb in BTN3-mediated negative signal transduction. The magnitude of the cell surface expression of BTN3 molecules correlated inversely with lymphocyte activity, suggesting that BTN3 molecules contribute to the maintenance of the immune system. Taken together, our findings provide new insights for the role of BTN3 as an inhibitor of excessive cellular immune responses.

In this study, we explored immune responses after intramuscular co-administration of the HIV-1 gp160 Env gene-expressing adenovirus (Ad) vector and modified vaccinia virus Ankara (MVA) vector in a mouse model. Surprisingly, the simultaneous vaccination of the two vaccines, either as a mixture or separately, suppressed responses, when compared with the administration of each vaccine separately. Ad vaccine or MVA vaccine, co-administered with a mock MVA or mock Ad vector, also resulted in suppressing HIV-specific effector T-cell responses, and a part of antigen-specific memory T-cell responses. In an in vitro experiment, the two vectors infected individual cells and MVA suppressed the transgene expression produced by the adenovirus vector. This viral interference may involve soluble factor(s), secreted by virus-infected cells. Our study may help in designing a vaccination regimen and in investigating viral interference.

オニグルミのアレロパシー活性がニセアカシアの初期生長に及ぼす効果を明らかにするために，混植実験と根圏土壌法による検定を行った。混植実験において，オニグルミとニセアカシアを混植した区では，対照区に比べて，ニセアカシアの乾物重量が約50％ に低下した。また， 混植区の土壌は，1.2％×10-6g g-1 のユグロンが含まれ，ニセアカシアの初期生長を50％ 阻害するユグロンの量とほぼ一致した。根圏土壌法による検定では，ニセアカシアの初期生長は根域土壌よりも根圏土壌で阻害される傾向を示し，根に近い土壌ほど生長が低下することから，根から出るユグロンが作用していることが強く示唆された。以上のことから，オニグルミが生育する土壌では，オニグルミの根のアレロパシー活性により，ニセアカシアの初期生長を阻害する可能性があることがわかった。

静岡県遠州灘海岸において，堆砂が海浜植生の成立に及ぼす影響を明らかにすることを目的に，植生と堆砂の経年変化を調査した。対象地の植生はTWINSPANによって3つの群落型に分類され，汀線から内陸に向かって，帯状に植生が変化した。DCAの序列化を行ったところ，1軸で各群落型は明瞭に分かれてプロットされた。また，1軸のスコアと堆砂深の間には，強い相関関係が認められた。このことから，各群落型の分布には，堆砂が影響していると考えられた。一方，群落型の経年変化を考慮に入れて解析したところ，各群落型の分布は，堆砂だけではなく，波浪や種子の供給など他の要因の影響を受けていると考えられた。

Background Adenovirus type 5 (Ad5) is widely used as a vehicle for vaccine delivery in the treatment of infectious disease and cancer. However, the efficacy of Ad5 vectors has been limited in humans because exposure to Ad5 infections results in most adults having neutralizing antibodies against Ad5. To overcome this limitation, the hexon epitope present in the fifth hypervariable region of Ad5 was modified. Methods To evaluate the ability of Ad5 vectors encoding the HIV env protein to induce Ag-specific immune responses in the face of pre-existing anti-Ad5 immunity, mice were administrated intramuscularly with the Ad-Luc vector, and then vaccinated with parental or hexon-modified Ad5 vectors (Ad-HisHIV, Ad-END/AAAHIV or Ad-HIV) at week 8. HIV-specific cell-mediated immune responses were detected through a combination of tetramer assays and intracellular cytokine staining from weeks 8–23. Results The hexon-modified Ad vector was able to escape from anti-Ad5 neutralizing antibody, and mice with the modified vector generated significantly lower individual neutralizing antibody than those immunized with the parental vector. Furthermore, mice with pre-existing anti-Ad immunity immunized with the modified vector generated significantly stronger cell-mediated anti-env responses than those immunized with the parental vector. Conclusions These data demonstrate that Ad5 vector with hexon modification reduce their sensitivity to pre-existing anti-Ad immunity and improve their clinical utility. Copyright © 2009 John Wiley & Sons, Ltd.

Background A monoclonal antibody (mAb) 2F5 binds to the membrane-proximal external region (MPER) of the transmembrane subunit gp41 of human immunodeficiency virus type 1 (HIV-1) is known to broadly neutralize HIV-1 strains. The Adenovirus type 5 vector (Ad5) has been widely applied for HIV-1 vaccine, and hexon hypervariable region 5 (HVR5) is exposed on viral surface and easily target host immune responses against Ad5. Methods We constructed a recombinant adenovirus type 5 vector (rAd5) with a 2F5-binding epitope (ELDKWA) of MPER on Ad5-HVR5. In addition, we developed rAd5 encoding the HIV-1IIIB envelope (Env) gene for the induction of Env-specific cellular immunity. Results The virus titers of the constructed rAd5 were similar to that of the parental Ad5 vector. Furthermore, high-dose immunization of rAd5 induced Env-specific CD8+ cells and high levels of anti-ELDKWA antibodies. Moreover, an in vitro HIV-1 neutralization assay indicated that ELDKWA-specific mAbs derived from rAd5-immunized mice neutralized a wide range of HIV-1 strains. Conclusions The present study outlines the development of an Ad5-based HIV-1 vaccine targeting the hypervariable regions of Ad5. The constructed rAd5 induced an HIV-1-specific cellular immune response and neutralizing antibodies against various strains of HIV-1 simultaneously. Copyright © 2008 John Wiley & Sons, Ltd.

本研究は，中国内陸部のテンゲル砂漠東端において，砂丘から扇状地末端の移行帯に成立する植生と微地形との対応関係を明らかにすることを目的とした。移行帯の植生は，砂丘から扇状地末端に向かって，Agriophyllum squarrosum→Bassia dasyphylla などアカザ科の種→Eragrostis poaeoides などイネ科やマメ科の種の順番で優占度が高くなった。また，移行帯の砂丘側で優占した A. squarrosum とArtemisia sphaerocephala の2 種は分布する立地が異なり，前者は砂丘斜面，後者は砂丘間低地を選好していると考えられた。これらのことから，移行帯では砂丘からの距離や微地形によって，成立する植生が異なることがわかった。

湘南海岸の新たに植生が定着した砂浜において，地形の変化及び植生の定着過程について調査を行った。対象地では汀線の前進，比高の上昇によって，波浪による攪乱が軽減され，植生の被覆面積が増加したことが示唆された。また7種の海浜植物の定着が確認されたが，長期間の海流散布が困難なオニシバなどの定着は確認されなかった。このことから，対象地では長期間の海流散布が困難な種の供給ポテンシャルが低いと推察された。更に植生と地形の関係について選好度指数を用いて解析したところ，出現種によって波浪強度が異なる立地を選好していた。以上のことから，人工養浜の際には，波浪強度が異なる立地環境を形成することが，海浜植生の定着において重要であると考えられた。