Enhancement of the antigen-specific cytotoxic T lymphocyte-inducing ability in the PMDC11 leukemic plasmacytoid dendritic cell line via lentiviral vector-mediated transduction of the caTLR4 gene.

The aim of the present study was to enhance the efficiency of leukemia immunotherapy by increasing the antigen-specific cytotoxic T lymphocyte-inducing ability of leukemia cells. The leukemic plasmacytoid dendritic cell line PMDC05 containing the HLA-A02/24 antigen, which was previously established in our laboratory (Laboratory of Hematology and Oncology, Graduate School of Health Sciences, Niigata University, Niigata, Japan), was used in the present study. It exhibited higher expression levels of CD80 following transduction with lentiviruses encoding the CD80 gene. This CD80-expressing PMDC05 was named PMDC11. In order to establish a more potent antigen-presenting cell for cellular immunotherapy of tumors or severe infections, PMDC11 cells were transduced with a constitutively active (ca) toll-like receptor 4 (TLR4) gene using the Tet-On system (caTLR4-PMDC11). CD8(+) T cells from healthy donors with HLA-A02 were co-cultured with mutant WT1 peptide-pulsed PMDC11, lipopolysaccharide (LPS)-stimulated PMDC11 or caTLR4-PMDC11 cells. Interleukin (IL)-2 (50 IU/ml) and IL-7 (10 ng/ml) were added on day three of culture. Priming with mutant WT1 peptide-pulsed PMDC11, LPS-stimulated PMDC11 or caTLR4-PMDC11 cells was conducted once per week and two thirds of the IL-2/IL-7 containing medium was replenished every 3-4 days. Immediately prior to the priming with these various PMDC11 cells, the cultured cells were analyzed for the secretion of interferon (IFN)-γ in addition to the percentage and number of CD8(+)/WT1 tetramer(+) T cells using flow cytometry. caTLR4-PMDC11 cells were observed to possess greater antigen-presenting abilities compared with those of PMDC11 or LPS-stimulated PMDC11 cells in a mixed leukocyte culture. CD8 T cells positive for the WT1 tetramer were generated following 3-4 weeks of culture and CD8(+)/WT1 tetramer+ T cells were markedly increased in caTLR4-PMDC11-primed CD8(+) T cell culture compared with PMDC11 or LPS-stimulated PMDC11-primed CD8(+) T cell culture. These CD8(+) T cells co-cultured with caTLR4-PMDC11 cells were demonstrated to secrete IFN-γ and to be cytotoxic to WT1-expressing target cells. These data suggested that the antigen-specific cytotoxic T lymphocyte (CTL)-inducing ability of PMDC11 was potentiated via transduction of the caTLR4 gene. The present study also suggested that caTLR4-PMDC11 cells may be applied as potent antigen-presenting cells for generating antigen-specific CTLs in adoptive cellular immunotherapy against tumors and severe viral infections

Dynamic action of an intrinsically disordered protein in DNA compaction that induces mycobacterial dormancy

抗酸菌における決定的な休眠誘導機構を発見 --天然変性タンパク質による新規のDNA凝集メカニズム--. 京都大学プレスリリース. 2024-02-14.Mycobacteria are the major human pathogens with the capacity to become dormant persisters. Mycobacterial DNA-binding protein 1 (MDP1), an abundant histone-like protein in dormant mycobacteria, induces dormancy phenotypes, e.g. chromosome compaction and growth suppression. For these functions, the polycationic intrinsically disordered region (IDR) is essential. However, the disordered property of IDR stands in the way of clarifying the molecular mechanism. Here we clarified the molecular and structural mechanism of DNA compaction by MDP1. Using high-speed atomic force microscopy, we observed that monomeric MDP1 bundles two adjacent DNA duplexes side-by-side via IDR. Combined with coarse-grained molecular dynamics simulation, we revealed the novel dynamic DNA cross-linking model of MDP1 in which a stretched IDR cross-links two DNA duplexes like double-sided tape. IDR is able to hijack HU function, resulting in the induction of strong mycobacterial growth arrest. This IDR-mediated reversible DNA cross-linking is a reasonable model for MDP1 suppression of the genomic function in the resuscitable non-replicating dormant mycobacteria

Association of blood pressure and renal outcome in patients with chronic kidney disease; a post hoc analysis of FROM-J study

It is well-known that hypertension exacerbates chronic kidney disease (CKD) progression, however, the optimal target blood pressure (BP) level in patients with CKD remains unclear. This study aimed to assess the optimal BP level for preventing CKD progression. The risk of renal outcome among different BP categories at baseline as well as 1 year after, were evaluated using individual CKD patient data aged between 40 and 74 years from FROM-J [Frontier of Renal Outcome Modifications in Japan] study. The renal outcome was defined as >= 40% reduction in estimated glomerular filtration rate to130 mmHg group. A significant increase in the renal outcome was found only in the group of diastolic BP >= 90 mmHg. The group of BP= 130 mmHg at baseline. Targeting SBP level<130 mmHg would be associated with the preferable renal outcome.Clinical Trial Registration-URL: https://www.umin.ac.jp/ctr/. Unique identifier: UMIN000001159 (16/05/2008)

Sex-inducing effects toward planarians widely present among parasitic flatworms

Summary Various parasitic flatworms infect vertebrates for sexual reproduction, often causing devastating diseases in their hosts. Consequently, flatworms are of great socioeconomic and biomedical importance. Although the cessation of parasitic flatworm sexual reproduction is a major target of anti-parasitic drug design, little is known regarding bioactive compounds controlling flatworm sexual maturation. Using the planarian Dugesia ryukyuensis, we observed that sex-inducing substances found in planarians are also widespread in parasitic flatworms, such as monogeneans and flukes (but not in tapeworms). Reverse-phase HPLC analysis revealed the sex-inducing substance(s) eluting around the tryptophan retention time in the fluke Calicophoron calicophorum, consistent with previous studies on the planarian Bipalium nobile, suggesting that the substance(s) is likely conserved among flatworms. Moreover, six of the 18 ovary-inducing substances identified via transcriptome and metabolome analyses are involved in purine metabolism. Our findings provide a basis for understanding and modifying the life cycles of various parasitic flatworms.journal articl

DOCK2 is involved in the host genetics and biology of severe COVID-19

「コロナ制圧タスクフォース」COVID-19疾患感受性遺伝子DOCK2の重症化機序を解明 --アジア最大のバイオレポジトリーでCOVID-19の治療標的を発見--. 京都大学プレスリリース. 2022-08-10.Identifying the host genetic factors underlying severe COVID-19 is an emerging challenge. Here we conducted a genome-wide association study (GWAS) involving 2, 393 cases of COVID-19 in a cohort of Japanese individuals collected during the initial waves of the pandemic, with 3, 289 unaffected controls. We identified a variant on chromosome 5 at 5q35 (rs60200309-A), close to the dedicator of cytokinesis 2 gene (DOCK2), which was associated with severe COVID-19 in patients less than 65 years of age. This risk allele was prevalent in East Asian individuals but rare in Europeans, highlighting the value of genome-wide association studies in non-European populations. RNA-sequencing analysis of 473 bulk peripheral blood samples identified decreased expression of DOCK2 associated with the risk allele in these younger patients. DOCK2 expression was suppressed in patients with severe cases of COVID-19. Single-cell RNA-sequencing analysis (n = 61 individuals) identified cell-type-specific downregulation of DOCK2 and a COVID-19-specific decreasing effect of the risk allele on DOCK2 expression in non-classical monocytes. Immunohistochemistry of lung specimens from patients with severe COVID-19 pneumonia showed suppressed DOCK2 expression. Moreover, inhibition of DOCK2 function with CPYPP increased the severity of pneumonia in a Syrian hamster model of SARS-CoV-2 infection, characterized by weight loss, lung oedema, enhanced viral loads, impaired macrophage recruitment and dysregulated type I interferon responses. We conclude that DOCK2 has an important role in the host immune response to SARS-CoV-2 infection and the development of severe COVID-19, and could be further explored as a potential biomarker and/or therapeutic target

Isolation and Analysis of Arabidopsis Mutants with Altered Leaf Thickness or Leaf Length

　Leaf shape in wild plants has diversely evolved from ancient times. A lot of researchers have challenged to elucidate mechanisms of the evolutional dynamics in plant leaf shape. Several studies carried out by using genetic model plants, such as Arabidopsis thaliana, Antirrhinum majus or Lycopersicon esculentum revealed a part of the genetic basis of variation in leaf shape. However, to get the general picture of evolutional dynamics in plant leaf shape, more studies are needed. We can divide things to three dimensions called length, width and height. Thus, he thought that three-dimensional classification of leaves would help to elucidate leaf-shape diversity. In previous studies, two classes of mutants, namely angustifolia (an) type and rotundifolia (rot) type with a specific alteration in either width or length of the leaf blade were identified in Arabidopsis. In both an and rot3 mutants, the leaf shape alteration is caused by the altered cell shape. However, the leaf dwarfism or the diversity of leaf shape is mainly caused by the variation of the cell number in wild plants. Therefore, he analyzed a dominant mutant, rotundifolia4-1D (rot4-1D), which possessed short leaves and floral organs caused by reduction of the cell number. On the other hand, variation of leaf thickness is mainly due to alteration of cell volume and/or cell number in wild plants. He have also isolated a mutant, working number N692, which possessed thick leaves caused by unusual cell expansion in the leaf thickness direction in Arabidopsis. 　In rot4-1D, anatomical observations showed that the altered leaf shape is caused by reduced cell proliferation specifically in the longtitudinal (proximal-distal) axis of the leaf, suggesting that the ROT4 gene controls polar cell proliferation in lateral organs. The ROT4 gene encodes a novel small peptide consisting of 53 amino acid residues. ROT4 open reading frame had not been identified in the Arabidopsis genome annotation. He cloned ROT4 full-length cDNA and registered it to the GenBank as accession number AB107209. ROT4 mRNA accumulates at a higher level in rot4-1D compared to wild type. Over-expression of a ROT4-green fluorescence protein (GFP) fusion protein in transgenic plants recapitulated the rot4-1D phenotype suggesting that ROT4 acts to restrict cell proliferation. The ROT4-GEP fusion protein localized to the plasma membrane when expressed in transgenic Arabidopsis plants. Database search and phylogenetic analysis indicate that ROT4 defines a novel, seed plant-specific family of small peptides with 22 members in Arabidopsis, ROT FOUR LIKE1-22 (RTFL1-22), and all RTFL members share a conserved 29 amino acid domain, the RTF domain. ROT4 contains N-terminal region (16 amino acid residues), RTF domain and C-terminal region (8 amino acid residues), and over-expression of the ROT4 truncated N-terminal region or C-terminal region was sufficient to confer a rot4-1D phenotype. Loss-of-function mutations in several RTFL genes were aphenotypic in Arabidopsis or Oryza sativa suggesting that there may be some functional redundancy among family members. RT-PCR analysis revealed that ROT4 is expressed in the shoot apex and young leaves of wild-type plants, consistent with a role for ROT4 in controlling polarity-dependent cell proliferation during wild-type leaf morphogenesis.　On the other hand, mutants with only changed leaf thickness had not been isolated. Therefore, as the first step to study thickness control in leaves, he developed an instrument, called Leaf Thickness Measurement Instrument (LTMI) that can measure the thickness of Arabidopsis living leaves reproducibly by a laser displacement sensor. The conventional measurement method requires several complicated and time consuming steps, such as fixation, embedding, slicing of the samples by microtome and finally observation under microscope. These steps can be omitted by using LTMI. Thick-leaved mutants were screened from a T-DNA activation-tagged library of C24 by using LTMI. By screening more than 2000 lines, he isolated three mutants N692, N865 and N091 with thicker or thinner leaves than wild type. He mainly analyzed N692 which has the most altered leaf thickness among the three mutants. The thickness of N692 leaves was 158 ± 10μm, while that of wild-type C24 leaves was 126 ± 6μm. In addition, leaves of N692 are tight, while those of wild type are waved and curly. Moreover, the stem of N692 is thicker than that of wild type. Anatomical analysis showed that palisade cells of N692 expand isotropically and number of the cells per leaf is reduced. As a result, the leaf blade area of N692 is equal to that of wild type. It is thought that compensation occurs in leaf area but not occurs in leaf thickness direction. A mutant which has such phenotype is unprecedent. 　Recently, movement of an application from outcome of model plants to properties of other wild plants has occurred in morphogenesis and related fields. In addition to an-like and rot-like mutants, by the isolation of N692, a description with three-dimensional geometry has been possible. It is thought analysis of seed-plant specific ROT4 and RTFL genes is suitable to apply for estimation of leaf shape in wild plants. Building on our previous success in the identification of AN and ROT3 genes, the present study of rot4-1D and N692 will possibly help to illustrate the variety of leaf shape in wild plants

Improved Prefrontal Activity and Chewing Performance as Function of Wearing Denture in Partially Edentulous Elderly Individuals: Functional Near-Infrared Spectroscopy Study

<div><p>The purpose of this study was to elucidate the effects of wearing a denture on prefrontal activity during chewing performance. We specifically examined that activity in 12 elderly edentulous subjects [63.1±6.1 years old (mean ± SD)] and 12 young healthy controls (22.1±2.3 years old) using functional near-infrared spectroscopy (fNIRS) in order to evaluate the quality of prefrontal functionality during chewing performance under the conditions of wearing a denture and tooth loss, and then compared the findings with those of young healthy controls. fNIRS and electromyography were used simultaneously to detect prefrontal and masticatory muscle activities during chewing, while occlusal force and masticatory score were also examined by use of a food intake questionnaire. A significant increase in prefrontal activity was observed during chewing while wearing a denture, which was accompanied by increased masticatory muscle activity, occlusal force, and masticatory score, as compared with the tooth loss condition. Prefrontal activation during chewing while wearing a denture in the elderly subjects was not much different from that in the young controls. In contrast, tooth loss in the elderly group resulted in marked prefrontal deactivation, accompanied by decreased masticatory muscle activity, occlusal force, and masticatory score, as compared with the young controls. We concluded that intrinsic prefrontal activation during chewing with a denture may prevent prefrontal depression induced by tooth loss in elderly edentulous patients.</p></div

Initial validations for pursuing irradiation using a gimbals tracking system.

Our newly designed image-guided radiotherapy (IGRT) system enables the dynamic tracking irradiation with a gimbaled X-ray head and a dual on-board kilovolt imaging subsystem for real-time target localization. Examinations using a computer-controlled three-dimensionally movable phantom demonstrated that our gimbals tracking system significantly reduced motion blurring effects in the dose distribution compared to the non-tracking state