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In this paper, we propose two peak-to-average power ratio (PAPR) reduction methods that apply adaptive in-band interference restriction based on singular value differences between spatial channels in uplink eigenmode massive multiple-input multiple-output (MIMO) transmission. In uplink transmission utilizing high-frequency bands, reducing PAPR is crucial to suppress non-linear distortion and ensure sufficient transmission distance, given the stringent amplification requirements of power amplifiers. Therefore, the proposed methods direct the PAPR reduction signals caused by clipping and filtering (CF) only to spatial channels with relatively small singular values, thereby avoiding in-band interference in spatial channels with larger singular values, which are more susceptible to such interference compared to the case without PAPR reduction. After that, the two proposed methods restrict PAPR reduction signals for all or a subset of subcarriers based on the transmission quality requirements. Both interference restrictions allow for more effective PAPR reduction while tolerating interference in the data streams. Computer simulations demonstrate that the transmission quality of the proposed methods is improved by up to approximately 30% compared to the case without PAPR reduction. Moreover, a comparative evaluation of the two proposed methods demonstrates that the interference restriction across all subcarriers effectively mitigates in-band interference caused by the resulting PAPR reduction signals while reducing the PAPR.

Abstract In chronic stages of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalitis (EAE), connexin (Cx)43 gap junction channel proteins are overexpressed because of astrogliosis. To elucidate the role of increased Cx43, the central nervous system (CNS)-permeable Cx blocker INI-0602 was therapeutically administered. C57BL6 mice with chronic EAE initiated by MOG35-55 received INI-0602 (40 mg/kg) or saline intraperitoneally every other day from days post-immunization (dpi) 17–50. Primary astroglia were employed to observe calcein efflux responses. In INI-0602-treated mice, EAE clinical signs improved significantly in the chronic phase, with reduced demyelination and decreased CD3+ T cells, Iba-1+ and F4/80+ microglia/macrophages, and C3+GFAP+ reactive astroglia infiltration in spinal cord lesions. Flow cytometry analysis of CD4+ T cells from CNS tissues revealed significantly reduced Th17 and Th17/Th1 cells (dpi 24) and Th1 cells (dpi 50). Multiplex array of cerebrospinal fluid showed significantly suppressed IL-6 and significantly increased IL-10 on dpi 24 in INI-0602-treated mice, and significantly suppressed IFN-γ and MCP-1 on dpi 50 in the same group. In vitro INI-0602 treatment inhibited ATP-induced calcium propagations of Cx43+/+ astroglial cells to similar levels of those of Cx43−/− cells. Astroglial Cx43 hemichannels represent a novel therapeutic target for chronic EAE and MS.

Abstract Conventional bivalent antibodies against cell surface receptors often initiate unwanted signal transduction by crosslinking two antigen molecules. Biparatopic antibodies (BpAbs) bind to two different epitopes on the same antigen, thus altering crosslinking ability. In this study, we develop BpAbs against tumor necrosis factor receptor 2 (TNFR2), which is an attractive immune checkpoint target. Using different pairs of antibody variable regions specific to topographically distinct TNFR2 epitopes, we successfully regulate the size of BpAb–TNFR2 immunocomplexes to result in controlled agonistic activities. Our series of results indicate that the relative positions of the two epitopes recognized by the BpAb are critical for controlling its signaling activity. One particular antagonist, Bp109-92, binds TNFR2 in a 1:1 manner without unwanted signal transduction, and its structural basis is determined using cryo-electron microscopy. This antagonist suppresses the proliferation of regulatory T cells expressing TNFR2. Therefore, the BpAb format would be useful in designing specific and distinct antibody functions.

The SARS-CoV-2 pandemic alerted the potential for significant harm due to future cross-species transmission of various animal coronaviruses to human. There is a significant need of antibody-based drugs to treat patients infected with previously unseen coronaviruses. In this study, we generated CV804, an antibody that binds to the S2 domain of SARS-CoV-2 spike protein, which is highly conserved across the coronavirus family and less susceptible to mutations. CV804 demonstrated broad cross-reactivities not only disease-associated human beta coronaviruses including SARS-CoV, MERS-CoV, HCoV-OC43, HCoV-HKU1 and with existing mutant strains of SARS-CoV-2 and but also with 20 representative animal-origin coronaviruses. CV804 exhibits strong antibody-dependent cellular cytotoxicity (ADCC) to SARS-CoV-2 spike protein expressed on cells in vitro, while completely lacks virus-neutralization activity. In animal models, CV804 suppressed disease progression caused by SARS-CoV-2 infection. Structural studies using HDX-MS combined with reactivity analysis with point mutants of recombinant spike proteins revealed that CV804 binds to a unique conformational epitope within the S2 domain of the spike proteins that is highly conserved among various coronaviruses. Overall, obtained data suggest that the non-neutralizing CV804 antibody recognizes the conformational structure of the spike protein displayed on the surface of infected cells and weakens the viral virulence by supporting the host immune cells' attack through ADCC activity in vivo. The CV804 epitope information revealed in this study is useful for designing pan-corona antibody therapeutics and universal coronavirus vaccines for preparing potential future pandemics.

We previously reported a novel secondary progressive multiple sclerosis (SPMS) model, progressive experimental autoimmune encephalomyelitis (pEAE), in oligodendroglia-specific Cx47-inducible conditional knockout (Cx47 icKO) mice. Based on our prior study showing the efficacy of iguratimod (IGU), an antirheumatic drug, for acute EAE treatment, we aimed to elucidate the effect of IGU on the SPMS animal model. We induced pEAE by immunizing Cx47 icKO mice with myelin oligodendrocyte glycoprotein peptide 35–55. IGU was orally administered from 17 to 50 days post-immunization. We also prepared a primary mixed glial cell culture and measured cytokine levels in the culture supernatant after stimulation with designated cytokines (IL-1α, C1q, TNF-α) and lipopolysaccharide. A migration assay was performed to evaluate the effect of IGU on the migration ability of T cells toward mixed glial cell cultures. IGU treatment ameliorated the clinical signs of pEAE, decreased the demyelinated area, and attenuated glial inflammation on immunohistochemical analysis. Additionally, IGU decreased the intrathecal IL-6 level and infiltrating Th17 cells. The migration assay revealed reduced Th17 cell migration and IL-6 levels in the culture supernatant after IGU treatment. Collectively, IGU successfully mitigated the clinical signs of pEAE by suppressing Th17 migration through inhibition of IL-6 production by proinflammatory-activated glial cells.

Endogenous ACE2 is a receptor for SARS-CoV-2 and a recombinant soluble ACE2 protein can inhibit SARS-CoV-2 infection acting as a decoy. Here the authors show that B38-CAP, an ACE2-like enzyme but not a decoy for the virus, is protective against SARS-CoV-2-induced lung injury in animal models.

Abstract A major class of bispecific antibodies (BsAbs) utilizes heterodimeric Fc to produce the native immunoglobulin G (IgG) structure. Because appropriate pairing of heavy and light chains is required, the design of BsAbs produced through recombination or reassembly of two separately-expressed antigen-binding fragments is advantageous. One such method uses intein-mediated protein trans-splicing (IMPTS) to produce an IgG1-based structure. An extra Cys residue is incorporated as a consensus sequence for IMPTS in successful examples, but this may lead to potential destabilization or disturbance of the assay system. In this study, we designed a BsAb linked by IMPTS, without the extra Cys residue. A BsAb binding to both TNFR2 and CD30 was successfully produced. Cleaved side product formation was inevitable, but it was minimized under the optimized conditions. The fine-tuned design is suitable for the production of IgG-like BsAb with high symmetry between the two antigen-binding fragments that is advantageous for screening BsAbs.

Abstract Background Brain astroglia are activated preceding the onset of experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis (MS). We characterized the effects of brain astroglia on spinal cord inflammation, focusing on astroglial connexin (Cx)43, because we recently reported that Cx43 has a critical role in regulating neuroinflammation. Methods Because glutamate aspartate transporter (GLAST)+ astroglia are enriched in the brain gray matter, we generated Cx43 fl/fl ;GLAST-CreER T2/+ mice that were brain gray matter astroglia-specific Cx43 conditional knockouts (Cx43 icKO). EAE was induced by immunization with myelin oligodendroglia glycoprotein (MOG) 35–55 peptide 10 days after tamoxifen injection. Cx43 fl/fl mice were used as controls. Results Acute and chronic EAE signs were significantly milder in Cx43 icKO mice than in controls whereas splenocyte MOG-specific responses were unaltered. Histologically, Cx43 icKO mice showed significantly less demyelination and fewer CD45+ infiltrating immunocytes, including F4/80+ macrophages, and Iba1+ microglia in the spinal cord than controls. Microarray analysis of the whole cerebellum revealed marked upregulation of anti-inflammatory A2-specific astroglia gene sets in the pre-immunized phase and decreased proinflammatory A1-specific and pan-reactive astroglial gene expression in the onset phase in Cx43 icKO mice compared with controls. Astroglia expressing C3, a representative A1 marker, were significantly decreased in the cerebrum, cerebellum, and spinal cord of Cx43 icKO mice compared with controls in the peak phase. Isolated Cx43 icKO spinal microglia showed more anti-inflammatory and less proinflammatory gene expression than control microglia in the pre-immunized phase. In particular, microglial expression of Ccl2, Ccl5, Ccl7, and Ccl8 in the pre-immunized phase and of Cxcl9 at the peak phase was lower in Cx43 icKO than in controls. Spinal microglia circularity was significantly lower in Cx43 icKO than in controls in the peak phase. Significantly lower interleukin (IL)-6, interferon-γ, and IL-10 levels were present in cerebrospinal fluid from Cx43 icKO mice in the onset phase compared with controls. Conclusions The ablation of Cx43 in brain gray matter astroglia attenuates EAE by promoting astroglia toward an anti-inflammatory phenotype and suppressing proinflammatory activation of spinal microglia partly through depressed cerebrospinal fluid proinflammatory cytokine/chemokine levels. Brain astroglial Cx43 might be a novel therapeutic target for MS.

COVID-19, caused by SARS-CoV-2, has spread worldwide with dire consequences. To urgently investigate the pathogenicity of COVID-19 and develop vaccines and therapeutics, animal models that are highly susceptible to SARS-CoV-2 infection are needed. In the present study, we established an animal model highly susceptible to SARS-CoV-2 via the intratracheal tract infection in CAG promoter–driven human angiotensin-converting enzyme 2–transgenic (CAG-hACE2) mice. The CAG-hACE2 mice showed several severe symptoms of SARS-CoV-2 infection, with definitive weight loss and subsequent death. Acute lung injury with elevated cytokine and chemokine levels was observed at an early stage of infection in CAG-hACE2 mice infected with SARS-CoV-2. Analysis of the hACE2 gene in CAG-hACE2 mice revealed that more than 15 copies of hACE2 genes were integrated in tandem into the mouse genome, supporting the high susceptibility to SARS-CoV-2. In the developed model, immunization with viral antigen or injection of plasma from immunized mice prevented body weight loss and lethality due to infection with SARS-CoV-2. These results indicate that a highly susceptible model of SARS-CoV-2 infection in CAG-hACE2 mice via the intratracheal tract is suitable for evaluating vaccines and therapeutic medicines.

Abstract Background Metastatic breast cancer (BCa) is most often diagnosed months after completion of treatment of the primary tumor when a patient reports physical symptoms. Besides a physical examination, no other alternative recurrence screening method is recommended for routine follow-up care. Detection of autoantibodies against tumor-associated antigens (TAAs) has demonstrated promise for distinguishing healthy women from patients diagnosed with primary BCa. However, it is unknown what changes occur to patient autoantibody levels during and after treatment. Methods Three serial blood draws were collected from 200 BCa patients: before treatment, 6 and 12 months after surgery. Patients were categorized according to treatment regimen, including surgery, chemotherapy, radiation, trastuzumab and hormonal therapies. The longitudinal samples were assayed for autoantibody responses against 32 conformation-carrying TAAs using a Luminex multiplex bead assay. Results The treatment modality groups that had the greatest decrease in autoantibody response levels were radiation + hormonal therapy; radiation + chemotherapy; and radiation + hormonal therapy + chemotherapy. For these three treatment groups, autoantibody responses against 9 TAAs (A1AT, ANGPTL4, CAPC, CST2, DKK1, GFRA1, GRN, LGALS3 and LRP10) were significantly reduced at 12 months after surgery compared to before treatment. One TAA, GRP78, had a significantly increased autoantibody response after 12 months. Conclusions Single treatment regimens alone did not significantly alter autoantibodies levels against the studied TAAs. Radiation treatment was the common denominator of the three most affected groups for significant changes in autoantibody response levels.

Malaria is a deadly infectious disease associated with fundamental changes in the composition of the memory B cell (MBC) compartment, most notably a large expansion of T-bet+ MBCs, termed atypical MBCs. However, we know little about the precursors of atypical MBCs and the conditions that drive their differentiation. We compared the responses of human tonsil naïve B cells, MBCs, and germinal center B cells to a variety of stimulatory conditions. We determined that prolonged antigen presentation in the presence of CpG and IFN-γ induced maximal expression of T-bet and other phenotypic markers of malaria-associated atypical MBCs primarily in naïve B cells in vitro. Importantly T-bet+ naïve-derived B cells resembled atypical MBCs in their hypo-responsiveness to signaling through their B cell receptors. Thus, naïve B cells can be induced to differentiate into phenotypically and functionally atypical-like MBCs in vitro under conditions that may prevail in chronic infectious diseases in vivo.

This work investigated the effects of co-occurring aflatoxin B1 (AFB1) and microcystin (MC) in aquaculture, using immunohistochemistry and genotoxicity methods. Tilapia (Oreochromis niloticus) were exposed to AFB1 by intraperitoneal and MC (cell extract of Microcystis aeruginosa) by intraperitoneal and immersion routes. The interaction of MC-AFB1 was evaluated co-exposing the intraperitoneal doses. Blood samples were collected after 8, 24, and 48h to analyze the micronucleus frequency and comet score. The interaction of MC-AFB1 showed a synergic mutagenic response by higher micronucleus frequency of co-exposed group. A slight genotoxic synergism was also observed in the comet score. Immunohistochemistry detected MC in al lthe fish liver tissues exposed to MC by intraperitoneal route, and only the immersed group with the highest dose of MC showed a positive response. Although MC was non-detectable in the edible muscle, the combination of immunohistochemistry with genotoxicity assay was an attractive biomonitoring tool in aquaculture, where the animals were frequently exposed to co-occurring synergic hazards.

The deterioration of the water quality due to aquaculture is associated with eutrophication, with bloom of cyanobacteria. Microcystis aeruginosa is distinguished as main producer of microcystins (MCs), group of hepatotoxins with tumor promoter potential. In the present work immunohistochemical method for detection of MC in tilápia (Oreochromis niloticus), fish submitted to intraperitoneal injection (i.p.) or immersion in extract of M. aeruginosa BCCBUSP 262 was developed, using monoclonal antibody anti - MC (M8H5) and polymer peroxidase system. The tilápias (N=42) had been submitted to the seven treatments, three groups inoculated i.p. with 2.0x105, 4.0x105 and 1.0x106 cells. Kg-1 of M. aeruginosa BCCBUSP 262 and four groups exposed to the immersion in different extract concentrations of cyanobacterium. Analyzing liver and muscular tissue for immunohistochemical assay, muscular tissue was not stained. All the animals inoculated i.p. presented positive marking for MC in the liver, but in immersion test, only the ones exposed in the highest dose (1,0x105 cels.mL-1) presented positive marking. Although MC was not detected in muscular tissue, as well as in the liver of animals immersed in extract of M. aeruginosa BCCBUSP 262 in concentrations less than 1.0x105 cels.mL-1, the results would constitute in the base for the methodological development aiming the application of the immunohistochemistry in the rapid diagnosis in quality control of fish.A deterioração da qualidade de água pela piscicultura associa-se à eutrofização, com florescimento de cianobactérias. Microcystis aeruginosa destaca-se como principal produtora de microcistinas (MCs), grupo de hepatotoxinas com potencial promotor de tumor. No presente trabalho desenvolveu-se método imunoistoquímico para a detecção de MC em tilápias (Oreochromis niloticus) submetidas à injeção intraperitoneal (i.p.) ou imersão em extrato de M. aeruginosa BCCBUSP 262, empregando anticorpo monoclonal anti-MC (M8H5) e sistema polímero-peroxidase. As tilápias (N=42) foram submetidas a sete tratamentos, sendo três grupos inoculados i.p. com 2,0x105, 4,0x105 e 1,0x106 cels.Kg-1 de M. aeruginosa BCCBUSP 262 e quatro submetidos à imersão em diferentes concentrações do extrato da cianobactéria (variando de 1,0x104 a 1,0x105cel.mL-1). Analisando fígado e tecido muscular pelo ensaio imunoistoquímico, não se detectou marcação em tecido muscular. Todos os animais inoculados i.p. apresentaram marcação positiva para MC no fígado, mas em teste de imersão, apenas os expostos a maior dose (1,0x105 cels.mL-1) apresentaram marcação positiva. Embora MC não seja detectada em tecido muscular, assim como no fígado de animais imersos em extrato de M. aeruginosa CCBUSP 262 em concentrações menores que 1,0x105 cels.mL-1, os resultados constituíram-se base para o desenvolvimento metodológico objetivando a aplicação da imunoistoquímica no diagnóstico rápido no controle de qualidade de pescados.