Biology subjects

Nakayama, M.

Publications and source records attributed to Nakayama, M..

31 records · Page 2Linked to original sources

bioRxiv2022

Fc-modified SARS-CoV-2 neutralizing antibodies with therapeutic effects in two animal models.

The use of therapeutic neutralizing antibodies against SARS-CoV-2 infection has been highly effective. However, there remain few practical antibodies against viruses that are acquiring mutations. In this study, we created 494 monoclonal antibodies from COVID-19-convalescent patients, and identified antibodies that exhibited comparable neutralizing ability to clinically used antibodies in the neutralization assay using pseudovirus and authentic virus including variants of concerns. These antibodies have different profiles against various mutations, which were confirmed by cell-based assay and cryo-electron microscopy. To prevent antibody-dependent enhancement, N297A modification was introduced, and showed a reduction of lung viral RNAs by therapeutic administration in a hamster model. In addition, an antibody cocktail consisting of three antibodies was also administered therapeutically to a macaque model, which resulted in reduced viral titers of swabs and lungs and reduced lung tissue damage scores. These results showed that our antibodies have sufficient antiviral activity as therapeutic candidates.

microbiology↗

bioRxiv2022

An attenuated vaccinia vaccine encoding the SARS-CoV-2 spike protein elicits broad and durable immune responses, and protects cynomolgus macaques and human ACE2 transgenic mice from SARS-CoV-2 and its variants

As long as the coronavirus disease 2019 (COVID-19) pandemic continues, new variants of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) with altered antigenicity will emerge. The development of vaccines that elicit robust, broad, and durable protection against SARS-CoV-2 variants is urgently needed. We have developed a vaccine (rDIs-S) consisting of the attenuated vaccinia virus DIs strain platform carrying the SARS-CoV-2 S gene. rDIs-S induced neutralizing antibody and T-lymphocyte responses in cynomolgus macaques and human angiotensin converting enzyme 2 (hACE2) transgenic mice, and showed broad protection against SARS-CoV-2 isolates ranging from the early-pandemic strain (WK-521) to the recent Omicron BA. 1 variant (TY38-839). Using a tandem mass tag (TMT) -based quantitative proteomic analysis of lung homogenates from hACE2 transgenic mice, we found that, among mice subjected to challenge infection with WK-521, vaccination with rDIs-S prevented protein expression related to the severe pathogenic effects of SARS-CoV-2 infection (tissue destruction, inflammation, coagulation, fibrosis, and angiogenesis) and restored protein expression related to immune responses (antigen presentation and cellular response to stress). Furthermore, long-term studies in mice showed that rDIs-S maintains S protein-specific antibody titers for at least 6 months after a 1st vaccination. Thus, rDIs-S appears to provide broad and durable protective immunity against SARS-CoV-2, including current and possibly future variants.

microbiology↗

bioRxiv2021

Basic reproduction numbers of three strains of mouse hepatitis viruses in mice

Mouse hepatitis virus (MHV) is a murine coronavirus and one of the most important pathogens in laboratory mice. Although various strains of MHV have been isolated, they are generally excreted in the feces and transmitted oronasally via aerosols and contaminated bedding. In this study, we attempted to determine the basic reproduction numbers of three strains of MHV to improve our understanding of MHV infections in mice. Five-week-old female C57BL/6J mice were inoculated intranasally with either the Y, NuU, or JHM variant strain of MHV and housed with two naive mice. After 4 weeks, the presence or absence of anti-MHV antibody in the mice was determined by the enzyme-linked immunosorbent assay. We also examined the distribution of MHV in the organs of Y, NuU, or JHM variant-infected mice. Our data suggest that the transmissibility of MHV is correlated with viral growth in the gastrointestinal tract of infected mice. To the best of our knowledge, this is the first report to address the basic reproduction numbers among pathogens in laboratory animals.

microbiology↗

bioRxiv2021

HLA class II mediates type 1 diabetes risk by anti-insulin repertoire selection

Type 1 diabetes (T1D) is a common autoimmune disorder characterized by the destruction of insulin-secreting pancreatic {beta} cells [1], in which polymorphism of the human leukocyte antigen (HLA) class II region is the major genetic risk factor [2, 3, 4]. However, how variation in class II molecules alters T1D risk remains a longstanding question. Here we show how T1D risk due to HLA class II haplotype combinations [5] correlates with the frequency of negatively charged sequences in the CDR3{beta} region of CD4+ T cell receptor (TCR) repertoires purified from peripheral blood. These sequences are known to be common in receptors that bind insulin B:9-23 [6], the primary autoantigen in T1D. We also show the same effect in circulating activated CD4+ T cells from newly-diagnosed T1D cases, and in islet-infiltrating T cells from patients with active T1D. Furthermore, we demonstrate that the proportion of insulin-reactive CD4+ T cells present in islets is predicted by the frequency of these negatively charged CDR3{beta} amino acid sequences. Our results suggest diagnostic uses of T cell repertoire profiling in early detection of insulin autoimmunity, and inform ongoing efforts to improve tolerance induction to insulin and prevention of T1D [7].

genomics↗

bioRxiv2021

NR4A3 is oxidative stress responsible transcription factor through HMOX1, and also controls cell cycle through CCNE1 and CDK2 in pancreatic islet derived 1.1B4 cells

The mechanism of antioxidant defense system is still controversial. As islet {beta}-cell is weak in oxidative condition, that causes diabetes mellitus, therefore, antioxidant defense system of human pancreatic islet derived 1.1B4 cell was analyzed. Cells were exposed to H2O2 and comprehensive gene expression was analyzed by Agilent human microarray. HMOX1 and NR4A3, member of orphan receptor, were up-regulated. Therefore, NR4A3 was knocked down with siRNA, then analyzed gene expression by microarray, and found that the knocked down cells were weak in oxidative stress. HMOX1 expression was strongly inhibited by siRNA of NR4A3, and NR4A3 responsible sequence of aaggtca was found near the HMOX1 gene, suggesting NR4A3 is oxidative stress responsible transcription factor through HMOX1 expression. The expression of CCNE1 and CDK2 was also inhibited by knocked down of NR4A3, it is suggested NR4A3 is also important transcription factor for cell growth regulation. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=146 SRC="FIGDIR/small/457070v1_ufig1.gif" ALT="Figure 1"> View larger version (32K): org.highwire.dtl.DTLVardef@18d0371org.highwire.dtl.DTLVardef@dd1eacorg.highwire.dtl.DTLVardef@108b1b9org.highwire.dtl.DTLVardef@1cbf899_HPS_FORMAT_FIGEXP M_FIG Hydrogen peroxide induces NR4A3 and binds to aaggtca sequence of HMOX1, and increased transcription of HMOX1. Resulting heme oxygenase produces biliverdin, antioxidants, from heme. NR4A3 also bind to aaggtca sequence of CDK2 and CCNE1, resulting CDK2 and Cyclin E. CDK2 bind to cyclin E and cell goes from G1 to S phase. C_FIG

genomics↗

bioRxiv2021

Optimization of an LNP-mRNA vaccine candidate targeting SARS-CoV-2 receptor-binding domain

In 2020, two mRNA-based vaccines, encoding the full length of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein, have been introduced for control of the coronavirus disease (COVID-19) pandemic1,2. However, reactogenicity, such as fever, caused by innate immune responses to the vaccine formulation remains to be improved. Here, we optimized a lipid nanoparticle (LNP)-based mRNA vaccine candidate, encoding the SARS-CoV-2 spike protein receptor-binding domain (LNP-mRNA-RBD), which showed improved immunogenicity by removing reactogenic materials from the vaccine formulation and protective potential against SARS-CoV-2 infection in cynomolgus macaques. LNP-mRNA-RBD induced robust antigen-specific B cells and follicular helper T cells in the BALB/c strain but not in the C57BL/6 strain; the two strains have contrasting abilities to induce type I interferon production by dendritic cells. Removal of reactogenic materials from original synthesized mRNA by HPLC reduced type I interferon (IFN) production by dendritic cells, which improved immunogenicity. Immunization of cynomolgus macaques with an LNP encapsulating HPLC-purified mRNA induced robust anti-RBD IgG in the plasma and in various mucosal areas, including airways, thereby conferring protection against SARS-CoV-2 infection. Therefore, fine-tuning the balance between the immunogenic and reactogenic activity of mRNA-based vaccine formulations may offer safer and more efficacious outcomes.

immunology↗

bioRxiv2020

Life cycle of Bacillus subtilis population: biofilm life cycle generation and response to environmental pH

Microbial populations are ubiquitous, many of which form robust, multicellular structures called biofilms that protect cells from environmental damage. Understanding their migration cycles is crucial for controlling target microbial populations. Here we develop a multilevel mathematical model that captures the migration cycle of Bacillus subtilis cell populations and reproduces the periodic expansion of colonies into concentric circles. We construct an input/output model that regulates cell types in response to environmental conditions; based on this, we propose a theoretical model for the generation of cell population migration cycles. We then construct a multilevel system that links the cell-level state regulation model with tissue-level partial differential equations governing cell density, signal, and resource fields. Through this approach, we successfully simulate cyclically expanding colonies as well as their environmental pH dependence. The proposed models provide a robust basis for describing highly self-regulating multicellular systems and predicting broader biofilm-related phenomena.

microbiology↗

bioRxiv2020

Neutralizing antibody-dependent and -independent immune responses against SARS-CoV-2 in cynomolgus macaques

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infectious disease (COVID-19) has been threatening the world because of severe symptoms and relatively high mortality. To develop vaccines and antiviral drugs for COVID-19, an animal model of SARS-CoV-2 infection is required to evaluate the efficacy of prophylactics and therapeutics in vivo. Therefore, we examined the pathogenicity of SARS-CoV-2 in cynomolgus macaques until 28 days after virus inoculation in the present study. Cynomolgus macaques showed body temperature rises after infection and X-ray radiographic viral pneumonia was observed in one of three macaques. However, none of the macaques showed life-threatening clinical signs of disease corresponding that approximately 80% of human patients did not show a critical disease in COVID-19. A neutralizing antibody against SARS-CoV-2 and T-lymphocytes that produced interferon (IFN)-{gamma} and interleukin (IL)-2 specifically for SARS-CoV-2 N protein were detected on day 14 in the macaque that showed viral pneumonia. On the other hand, in the other macaques, in which a neutralizing antibody was not detected, T-lymphocytes that produced IFN-{gamma} specifically for SARS-CoV-2 N protein increased on day 7 to day 14 prior to an increase in the number of T-lymphocytes that produced IL-2. These results suggest that not only a neutralizing antibody but also cellular immunity augmented by IFN-{gamma} has a role in the elimination of SARS-CoV-2. Thus, because of the mild clinical signs of disease and low/no antibody responses against SARS-CoV-2 in two thirds of the macaques, cynomolgus macaques are appropriate to extrapolate human responses in vaccine and drug development. Author SummarySevere acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infectious disease (COVID-19) has been threatening the world. To develop vaccines and antiviral drugs for COVID-19, an animal model of SARS-CoV-2 infection is required to evaluate their efficacy in vivo. Therefore, we examined the pathogenicity of SARS-CoV-2 in a non-human primate model until 28 days after virus inoculation. Cynomolgus macaques showed a fever after infection and X-ray radiographic viral pneumonia was observed in one of three macaques. However, none of the macaques showed life-threatening symptoms. A neutralizing antibody against SARS-CoV-2 and T-lymphocytes that produced interferon (IFN)-{gamma} and interleukin (IL)-2 specifically for SARS-CoV-2 protein were detected on day 14 in the macaque that showed viral pneumonia. In the other macaques, in which a neutralizing antibody was not detected, T-lymphocytes that produced IFN-{gamma} specifically for SARS-CoV-2 N protein increased on day 7 to day 14. These results suggest that not only a neutralizing antibody but also cellular immunity augmented by IFN-{gamma} has a role in the elimination of SARS-CoV-2. Thus, because of the mild symptoms and low/no antibody responses against SARS-CoV-2 in two thirds of the macaques, cynomolgus macaques are appropriate to extrapolate human responses in vaccine and drug development.

microbiology↗

bioRxiv2020

Interleukin-11 is a Marker for Both Cancer- and Inflammation-Associated Fibroblasts that Contribute to Colorectal Cancer Progression

Interleukin (IL)-11 is a member of the IL-6 family of cytokines and involved in multiple cellular responses, including tumor development. However, the origin and functions of IL-11-producing (IL-11+) cells are not fully understood. To characterize IL-11+ cells in vivo, we generated Il11 reporter mice. IL-11+ cells appeared in the colon of three murine tumor models, and a murine acute colitis model. Il11ra1 or Il11 deletion attenuated the development of colitis-associated colorectal cancer. IL-11+ cells expressed fibroblast markers, and genes associated with cell proliferation and tissue repair. IL-11 induced STAT3 phosphorylation in colonic fibroblasts, suggesting the activation of IL-11+ fibroblasts. Analysis using the human cancer database revealed that genes enriched in IL-11+ fibroblasts were elevated in human colorectal cancer, and correlated with reduced disease-free survival. Together, our results suggested that tumor cells induced IL-11+ fibroblasts, and that a feed-forward loop between IL-11 and IL-11+ fibroblasts might contribute to tumor development.

cancer biology↗

bioRxiv2019

ADCK4 deficiency destabilizes the coenzyme Q complex, which is rescued by 2,4-dihydroxybenzoic acid treatment

ADCK4 mutations usually manifest as steroid-resistant nephrotic syndrome, and cause coenzyme Q10 (CoQ10) deficiency. However, the function of ADCK4 remains obscure. We investigated ADCK4 function using mouse and cell models. Podocyte-specific Adck4 deletion in mice significantly reduced survival and caused severe focal segmental glomerular sclerosis with extensive interstitial fibrosis and tubular atrophy, which were prevented by treatment with 2,4-dihydroxybenzoic acid (2,4-diHB), an analog of CoQ10 precursor molecule. ADCK4 knockout podocytes exhibited significantly decreased CoQ10 level, respiratory chain activity, mitochondrial potential, and dysmorphic mitochondria with loss of cristae formation, which were rescued by 2,4-diHB treatment, thus attributing these phenotypes to decreased CoQ10 levels. ADCK4 interacted with mitochondrial proteins including COQ5, and also cytoplasmic proteins including myosin and heat shock proteins. ADCK4 knockout decreased COQ complex levels, and the COQ5 level was rescued by ADCK4 overexpression in ADCK4 knockout podocytes. Overall, ADCK4 is required for CoQ10 biosynthesis and mitochondrial function in podocytes.

genetics↗

bioRxiv2019

KDM2 proteins constrain transcription from CpG island gene promoters independently of their histone demethylase activity

CpG islands (CGI) are associated with the majority of mammalian gene promoters and function to recruit chromatin modifying enzymes. It has therefore been proposed that CGIs regulate gene expression through chromatin-based mechanisms, however in most cases this has not been directly tested. Here, we reveal that the histone H3 lysine 36 (H3K36) demethylase activity of the CGI-binding KDM2 proteins contributes only modestly to the H3K36me2-depleted state at CGI-associated gene promoters and is dispensable for normal gene expression. Instead, we discover that KDM2 proteins play a widespread and demethylase-independent role in constraining gene expression from CGI-associated gene promoters. We further show that KDM2 proteins shape RNA Polymerase II occupancy but not chromatin accessibility at CGI-associated promoters. Together this reveals a demethylase-independent role for KDM2 proteins in transcriptional repression and uncovers a new function for CGIs in constraining gene expression.

molecular biology↗

bioRxiv2018

FBXL19 recruits CDK-Mediator to CpG islands of developmental genes to prime them for activation during lineage commitment

CpG islands are gene regulatory elements associated with the majority of mammalian promoters, yet how they regulate gene expression remains poorly understood. Here, we identify FBXL19 as a CpG island-binding protein in mouse embryonic stem (ES) cells and show that it associates with the CDK-Mediator complex. We discover that FBXL19 recruits CDK-Mediator to CpG island-associated promoters of non-transcribed developmental genes to prime these genes for activation during cell lineage commitment. We further show that recognition of CpG islands by FBXL19 is essential for mouse development. Together this reveals a new CpG island-centric mechanism for CDK-Mediator recruitment to developmental gene promoters in ES cells and a requirement for CDK-Mediator in priming these developmental genes for activation during cell lineage commitment.

developmental biology↗

bioRxiv2017

The somatically generated T cell receptor CDR3a contributes to the MHC allele specificity of the T cell receptor

Mature T cells bearing {beta} T cell receptors react with foreign antigens bound to alleles of major histocompatibility complex proteins (MHC) that they were exposed to during their development in the thymus, a phenomenon known as positive selection. The structural basis for positive selection has long been debated. Here, using mice expressing one of two different T cell receptor {beta} chains and various MHC alleles, we show that positive selection-induced MHC bias of T cell receptors is affected both by the germline encoded elements of the T cell receptor and {beta} chain and, surprisingly, dramatically affected by the non germ line encoded CDR3 of the T cell receptor chain. Thus, in addition to determining specificity for antigen, the non germline encoded elements of T cell receptors may help the proteins cope with the extremely polymorphic nature of major histocompatibility complex products within the species.

immunology↗