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The recent emergence of SARS-CoV-2 Omicron (B.1.1.529 lineage) variants possessing numerous mutations has raised concerns of decreased effectiveness of current vaccines, therapeutic monoclonal antibodies and antiviral drugs for COVID-19 against these variants1,2. The original Omicron lineage, BA.1, prevailed in many countries, but more recently, BA.2 has become dominant in at least 68 countries3. Here we evaluated the replicative ability and pathogenicity of authentic infectious BA.2 isolates in immunocompetent and human ACE2-expressing mice and hamsters. In contrast to recent data with chimeric, recombinant SARS-CoV-2 strains expressing the spike proteins of BA.1 and BA.2 on an ancestral WK-521 backbone4, we observed similar infectivity and pathogenicity in mice and hamsters for BA.2 and BA.1, and less pathogenicity compared with early SARS-CoV-2 strains. We also observed a marked and significant reduction in the neutralizing activity of plasma from individuals who had recovered from COVID-19 and vaccine recipients against BA.2 compared to ancestral and Delta variant strains. In addition, we found that some therapeutic monoclonal antibodies (REGN10987 plus REGN10933, COV2-2196 plus COV2-2130, and S309) and antiviral drugs (molnupiravir, nirmatrelvir and S-217622) can restrict viral infection in the respiratory organs of BA.2-infected hamsters. These findings suggest that the replication and pathogenicity of BA.2 is similar to that of BA.1 in rodents and that several therapeutic monoclonal antibodies and antiviral compounds are effective against Omicron BA.2 variants.

To establish a circular manufacturing system, continuous management of the resource flow and the condition of individual items including products, components, and the materials in the system is indispensable. This study proposes a life cycle simulation (LCS) based management method to support optimal decision-making under conditions that include various uncertainties, such as consumer behavior and market trends. LCS is a simulation method that simulates dynamic material flow in the entire life cycle system and enables the evaluation of environmental and economic efficiencies. Since the simulation model consists of many parameters, exhaustive simulations with all parameter combinations are impossible in a practical timeframe. Therefore, the method extracts parameters that have a significant impact on eco-efficiency through sensitivity analysis using LCS. Then, a response surface of multi-level resolution is created in a multidimensional space composed of the parameters. The response surface is used to quickly identify optimal parameter values under uncertain conditions for effective management. As a case study, the proposed method is applied to the management of a circular manufacturing system for lithium-ion batteries of automotive to verify the effectiveness of the management on eco-efficiency.

The BA.2 sublineage of the SARS-CoV-2 Omicron variant has become dominant in most countries around the world; however, the prevalence of BA.4 and BA.5 is increasing rapidly in several regions. BA.2 is less pathogenic in animal models than previously circulating variants of concern1–4. Compared with BA.2, however, BA.4 and BA.5 possess additional substitutions in the spike protein, which play a key role in viral entry, raising concerns that the replication capacity and pathogenicity of BA.4 and BA.5 are higher than those of BA.2. Here we have evaluated the replicative ability and pathogenicity of BA.4 and BA.5 isolates in wild-type Syrian hamsters, human ACE2 (hACE2) transgenic hamsters and hACE2 transgenic mice. We have observed no obvious differences among BA.2, BA.4 and BA.5 isolates in growth ability or pathogenicity in rodent models, and less pathogenicity compared to a previously circulating Delta (B.1.617.2 lineage) isolate. In addition, in vivo competition experiments revealed that BA.5 outcompeted BA.2 in hamsters, whereas BA.4 and BA.2 exhibited similar fitness. These findings suggest that BA.4 and BA.5 clinical isolates have similar pathogenicity to BA.2 in rodents and that BA.5 possesses viral fitness superior to that of BA.2.

During the current coronavirus disease 2019 (COVID-19) pandemic, a variety of mutations have accumulated in the viral genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and, at the time of writing, four variants of concern are considered to be potentially hazardous to human society1. The recently emerged B.1.617.2/Delta variant of concern is closely associated with the COVID-19 surge that occurred in India in the spring of 2021 (ref. 2). However, the virological properties of B.1.617.2/Delta remain unclear. Here we show that the B.1.617.2/Delta variant is highly fusogenic and notably more pathogenic than prototypic SARS-CoV-2 in infected hamsters. The P681R mutation in the spike protein, which is highly conserved in this lineage, facilitates cleavage of the spike protein and enhances viral fusogenicity. Moreover, we demonstrate that the P681R-bearing virus exhibits higher pathogenicity compared with its parental virus. Our data suggest that the P681R mutation is a hallmark of the virological phenotype of the B.1.617.2/Delta variant and is associated with enhanced pathogenicity.

The recent emergence of B.1.1.529, the Omicron variant1,2, has raised concerns of escape from protection by vaccines and therapeutic antibodies. A key test for potential countermeasures against B.1.1.529 is their activity in preclinical rodent models of respiratory tract disease. Here, using the collaborative network of the SARS-CoV-2 Assessment of Viral Evolution (SAVE) programme of the National Institute of Allergy and Infectious Diseases (NIAID), we evaluated the ability of several B.1.1.529 isolates to cause infection and disease in immunocompetent and human ACE2 (hACE2)-expressing mice and hamsters. Despite modelling data indicating that B.1.1.529 spike can bind more avidly to mouse ACE2 (refs. 3,4), we observed less infection by B.1.1.529 in 129, C57BL/6, BALB/c and K18-hACE2 transgenic mice than by previous SARS-CoV-2 variants, with limited weight loss and lower viral burden in the upper and lower respiratory tracts. In wild-type and hACE2 transgenic hamsters, lung infection, clinical disease and pathology with B.1.1.529 were also milder than with historical isolates or other SARS-CoV-2 variants of concern. Overall, experiments from the SAVE/NIAID network with several B.1.1.529 isolates demonstrate attenuated lung disease in rodents, which parallels preliminary human clinical data.

EG.5.1 is a subvariant of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Omicron XBB variant that is rapidly increasing in prevalence worldwide. However, the pathogenicity, transmissibility, and immune evasion properties of isolates of EG.5.1 are largely unknown. Here, we show that there are no obvious differences in growth ability and pathogenicity between EG.5.1 and XBB.1.5 in hamsters. We also demonstrate that, like XBB.1.5, EG.5.1 is transmitted more efficiently between hamsters compared to its predecessor, BA.2. In contrast, unlike XBB.1.5, we detect EG.5.1 in the lungs of four of six exposed hamsters, suggesting that the virus properties of EG.5.1 are different from those of XBB.1.5. Finally, we find that the neutralizing activity of plasma from convalescent individuals against EG.5.1 was slightly, but significantly, lower than that against XBB.1.5 or XBB.1.9.2. Our data suggest that the different virus properties after transmission and the altered antigenicity of EG.5.1 may be driving its increasing prevalence over XBB.1.5 in humans.

In peritoneal dialysis (PD) therapy, physical stresses such as exposure to peritoneal dialysate, catheter trauma, and peritonitis may induce peritoneal injury that can prevent continued long-term PD therapy. Therefore, protection of the peritoneum is an important target to enable long-term PD therapy in patients with end-stage renal disease. We previously showed that neutralization of the membrane complement regulators (CRegs) Crry and CD59 in rat peritoneum provokes development of acute peritoneal injury due to uncontrolled complement activation. C5a is a key effecter molecule of the complement system released during acute inflammation. Control of C5a has been proposed as a strategy to suppress inflammatory reactions and, because peritoneal injury is accompanied by inflammation, we hypothesized that C5a targeted therapy might be an effective way to suppress peritoneal injury. In the present study we used an established acute peritonitis model induced by neutralization of CRegs to investigate the effects on acute peritoneal injury of inhibiting C5a. Intravenous administration of an anti-C5a complementary peptide (AcPepA) up to 4 h after induction of injury significantly and dose-dependently prevented accumulation of inflammatory cells and reduced tissue damage in the model, accompanied by decreased C3b deposition. We show that C5a contributed to the development of peritoneal injury. Our results suggest that C5a is a target for preventing or treating peritoneal injury in patients undergoing prolonged PD therapy or with infectious complications.

Background:Oxidative stress (oxidant–antioxidant imbalance) plays an important role in the pathophysiology of neonatal sepsis. This study evaluated whether an antisense peptide endothelin receptor antagonist, ETR-P1/fl, could attenuate oxidative stress in a neonatal sepsis model.Methods:A total of 18 3-d-old piglets were anesthetized and mechanically ventilated. Six piglets received cecal ligation and perforation (CLP group) for induction of sepsis. Six piglets also received continuous infusion (0.05?mg/kg/h) of ETR-P1/fl 30?min after CLP (ETR-P1/fl group). Six piglets received a sham operation. Serum total hydroperoxide (TH), biological antioxidant potentials (BAPs), oxidative stress index (OSI, calculated as TH/BAP), interleukin (IL)-6, serum glutamic oxaloacetic transaminase (GOT), and creatinine were measured before CLP and at 1, 3, and 6?h after CLP.Results:CLP evoked a state of shock resulting in elevated TH, OSI, and IL-6 levels. ETR-P1/fl administration after CLP resulted in lower serum TH at 1 and 3?h after CLP, OSI at 1 and 3?h after CLP, IL-6 at 1 and 3?h after CLP, and GOT at 3 and 6?h after CLP as compared with the CLP group.Conclusion:ETR-P1/fl treatment significantly attenuated the elevation of serum oxidative stress markers (TH and OSI), IL-6, and GOT in a progressive neonatal sepsis CLP model.

Peritoneal fibrosis (PF) is an important complication of peritoneal dialysis (PD) therapy that often occurs in association with peritoneal high transport rate and ultrafiltration failure (UFF). To study the possible pathogenic role of connective tissue growth factor (CTGF) in the relationship of PF and UFF, dialysate CTGF contents (n= 178) and tissue CTGF expression (n= 61) were investigated by ELISA, real-time PCR, immunohistochemistry, and in situ hybridization. CTGF production with and without TGF-β1stimulation in human peritoneal mesothelial cells (HPMC) from the spent patients' peritoneal dialysate (n= 32) was studied in vitro. The dialysate-to-plasma ratio for creatinine (D/P Cr) was positively correlated to dialysate CTGF concentration and estimated local peritoneal production of CTGF. CTGF mRNA expression was 11.4-fold higher in peritoneal membranes with UFF than in pre-PD renal failure peritoneum and was correlated with thickness of the peritoneum. CTGF protein and mRNA were detected in mesothelium and in fibroblast-like cells. In cultured HPMC, TGF-β1-induced expression of CTGF mRNA was increased at 12 and 24 h and was correlated with D/P Cr. In contrast, bone morphogenic protein-4 mRNA expression was inversely correlated with D/P Cr. Our results suggest that high peritoneal transport state is associated with fibrosis and increased peritoneal CTGF expression and production by mesothelial cells, which can be stimulated by TGF-β1. Dialysate CTGF concentration could be a biomarker for both peritoneal fibrosis and membrane function. Functional alteration of mesothelial cells may be involved in progression of peritoneal fibrosis in high transport state.

The SARS-CoV-2 B.1.621 (Mu) variant emerged in January 2021 and was categorized as a variant of interest by the World Health Organization in August 2021. This designation prompted us to study the sensitivity of this variant to antibody neutralization. In a live virus neutralization assay with serum samples from individuals vaccinated with the Pfizer/BioNTech or Moderna mRNA vaccines, we measured neutralization antibody titers against B.1.621, an early isolate (spike 614D), and a variant of concern (B.1.351, beta variant). We observed reduced neutralizing antibody titers against the B.1.621 variant (3.4 to 7-fold reduction, depending on the serum sample and time after the second vaccination) compared to the early isolate and a similar reduction when compared to B.1.351. Likewise, convalescent serum from hamsters previously infected with an early isolate neutralized B.1.621 to a lower degree. Despite this antibody titer reduction, hamsters could not be efficiently re-challenged with the B.1.621 variant, suggesting the immune response to the first infection is adequate to provide protection against a subsequent infection with the B.1.621 variant.

Host selective toxins (HSTs) are small molecule phytotoxins that control the pathogenicity of microbes in the host plant, but the mechanistic basis for their selectivity is unknown. We developed AcIle-EDA (Aclle-(+)-9,10-epoxy-8-hydroxy-9-methyldeca-trienoic acid) as a molecular probe of an HST, examined its mode of action in genetically modified Oryza sativa, and found it to trigger ROS production through NADPH-oxidase OsRBOHB, causing the emergence of pathogenic traits. This result strongly suggests that AcIle-EDA functions through the hijacking of the plant–microbe interaction system.

The phosphatidylcholine-using phospholipase D (PLD) isoform PLD2 is widely expressed in mammalian cells and is activated in response to a variety of promitogenic agonists. In this study, active and inactive hemagglutinin-tagged human PLD2 (HA-PLD2) constructs were stably expressed in an EL4 cell line lacking detectable endogenous PLD1 or PLD2. The overall goal of the study was to examine the roles of PLD2 in cellular signal transduction and cell phenotype. HA-PLD2 confers PLD activity that is activated by phorbol ester, ionomycin, and okadaic acid. Proliferation and Erk activation are unchanged in cells transfected with active PLD2; proliferation rate is decreased in cells expressing inactive PLD2. Basal tyrosine phosphorylation of focal adhesion kinase (FAK) is increased in cells expressing active PLD2, as is phosphorylation of Akt; inactive PLD2 has no effect. Expression of active PLD2 is associated with increased spreading and elongation of cells on tissue culture plastic, whereas inactive PLD2 inhibits cell spreading. Inactive PLD2 also inhibits cell adhesion, migration, and serum-induced invasion. Cells expressing active PLD2 form metastases in syngeneic mice, as do the parental cells; cells expressing inactive PLD2 form fewer metastases than parental cells. In summary, active PLD2 enhances FAK phosphorylation, Akt activation, and cell invasion in EL4 lymphoma cells, whereas inactive PLD2 exerts inhibitory effects on adhesion, migration, invasion, and tumor formation. Overall, expression of active PLD2 enhances processes favorable to lymphoma cell metastasis, whereas expression of inactive PLD2 inhibits metastasis.

Crry is a rodent membrane–bound inhibitor of complement activation and is a structural and functional analog of the human complement inhibitors decay-accelerating factor and membrane cofactor protein. We found previously that expression of rat Crry on a human tumor cell line enhances tumorigenicity in nude rats. In this study, we investigated the effect that rat Crry expressed on tumor cells has on rat cell–mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity (ADCC). The expression of rat Crry on the surface of different human tumor cell lines inhibited ADCC mediated by rat natural killer (NK) cells. C3 opsonization is known to enhance NK cell–mediated cytolysis, and a potential mechanism for Crry-mediated inhibition of NK cell lysis is through Crry modulation of C3 deposition on target cells. However, the transfection of tumor cell lines with Crry enhanced their resistance to NK cell–mediated lysis in the absence of exogenous complement. The resistance of Crry-expressing tumor cells to NK cell–mediated ADCC could be reversed by treatment with anti–Crry F(ab)2. In addition, anti–Crry F(ab)2 enhanced the susceptibility of 13762 rat mammary adenocarcinoma cells (that endogenously express Crry) to ADCC mediated by allogeneic rat NK cells in the absence of added complement. We found no evidence that rat NK cells were a source of complement for target cell deposition during the in vitro cytolysis assay. These data suggest a novel function for rat Crry in tumor immune surveillance that may be unrelated to complement inhibition.

Activation of the complement system on the cell surface results in the insertion of pore forming membrane attack complexes (MAC, C5b‐9). In order to protect themselves from the complement attack, the cells express several regulatory molecules, including the terminal complex regulator CD59 that inhibits assembly of the large MACs by inhibiting the insertion of additional C9 molecules into the C5b‐9 complex. Using the whole cell patch clamp method, we were able to measure accumulation of homologous MACs in the membrane of CD59−human B‐cells, which formed non‐selective ion channels with a total conductance of 360 ± 24 pS as measured at the beginning of the steady‐state phase of the inward currents. C5b‐8 and small‐size MAC (MAC containing only a single C9) can also form ion channels. Nevertheless, in CD59+human B‐cells in spite of small‐size MAC formation, an ion current could not be detected. In addition, restoring CD59 to the membrane of the CD59−cells inhibited the serum‐evoked inward current. The ion channels formed by the small‐size MAC were therefore sealed, indicating that CD59 directly interfered with the pore formation of C5b‐8 as well as that of small‐size C5b‐9. These results offer an explanation as to why CD59‐expressing cells are not leaky in spite of a buildup of homologous C5b‐8 and small‐size MAC. Our experiments also confirmed that ion channel inhibition by CD59 is subject to homologous restriction and that CD59 cannot block the conductivity of MAC when generated by xenogenic (rabbit) serum.

Serum macromolecules are transported through the vascular endothelial layer to the interstitium via the caveolae and interendothelial clefts, but the nature of the permeability of these structures is unknown, and the manner of caveola‐vesicle transport is controversial. We have developed a method of detecting macromolecular channels using an in situ HRP perfusion into arteries previously perfused with aldehyde and random conventional sectioning for electron microscopy. Using unbiased morphometry, 4.75% of the abluminal caveolae and 15.13% of the intercellular clefts were the tracer‐positive in rat aortic endothelium. In rat aortas treated with N‐ethylmaleimide, all caveolae and most free vesicles in the cytoplasm except those around the Golgi area were HRP‐positive in the endothelial cells; 1.48% of abluminal caveolae were structurally recognized as caveolar channels through the endothelial layer in a plane of single section. The length density of the abluminal caveolae was decreased to about 80% to the physiological control level whereas the larger invaginations were more frequently observed. Moreover 96.17% of the intercellular clefts were HRP‐positive. We suggest that a flexible channel‐system functions extensively as a macromolecular transport pathway in the arterial endothelium in vivo because the tracer‐labeled abluminal caveolae and intercellular clefts should be opened to the luminal surfaces methodologically. We therefore propose that caveolar channels, rather than transcytosis, provide a mechanism of caveola‐vesicle transport in the endothelial cells, because free vesicles involved in transcytosis were few in number. Anat Rec 264:32–42, 2001. © 2001 Wiley‐Liss, Inc.

Antisense amino acids are amino acids which can be translated from the corresponding anti‐codons of a sense amino acid. Antisense peptides encoded by the noncoding DNA strand have a tendency to interact with each other. We have demonstrated that antisense peptide sequences are present intramolecularly, and these may contribute to the folding and maintenance of the tertiary structure of a protein. T20 is a synthetic peptide with an amino acid sequence in the gp41 of HIV‐1 and has been demonstrated to be a potent inhibitor of HIV‐1 infection. We searched for intramolecular peptide sequences which are antisense to portions of T20. A synthetic peptide (TA‐1L) consisting of amino acids 84 to 97 of gp160, which contains an antisense peptide sequence (TA‐1) to T20, was shown to inhibit HIV‐1mBinfection of MT‐4 cells. Interaction of these antisense peptides could be involved in sustaining HIV‐1 infectivity. The TA‐1L site, which exists in the C1 domain of gp160, is highly homologous among strains of HIV‐1, especially at TA‐1 and in the amino acids flanking the C terminus. Although the TA‐1 sites of 18 out of 30 HIV‐1 strains were antisense to the T20 region, those of the remaining 12 strains, including HIV‐1MN, were not. However, TA‐1L inhibited infection by HIV‐1MN, which has no antisense peptide in T20 corresponding to TA‐1, although the inhibitory effect was weaker. TA‐1L may thus also interfere with the gp160 interaction with CD4, which has an antisense sequence to TA‐1.

Several membrane proteins prevent host cells from homologous complement attack. In humans, one such protein, decay‐accelerating factor (DAF), exists as two isoforms, a GPI anchored form and a secreted form, which are generated by alternative splicing. DAF in mouse is also expressed as two isoforms, a GPI anchored form (GPI‐DAF) and a transmembrane form (TM‐DAF), which are produced from two separate genes. In this study, we transfected cDNA of mouse GPI‐DAF or TM‐DAF into Chinese hamster ovary (CHO) cells. Both isoforms of DAF on CHO cells were shown to regulate mouse complement C3 deposition mediated by the classical and alternative pathways and the inhibitory activity of both isoforms was species restricted. The two mouse DAF isoforms were effective against rat complement but not against human and guinea pig complement. Furthermore, we produced hamster mAbs to mouse DAF using GPI‐DAF transfectant cells and established seven unique mAbs (RIKO‐1–7). Western blotting analysis using RIKO‐3, which reacts with both GPI‐DAF and TM‐DAF, and RIKO‐4, which is an anti‐GPI‐DAF specific mAb, indicated that GPI‐DAF was expressed on erythrocytes, spleen and testis, and that TM‐DAF was expressed only in testis.

The HA protein of influenza A viruses is the major viral antigen. In this study, we simultaneously introduced mutations at 17 amino acid positions of an H5 HA expected to affect antigenicity. Viruses with ≥13 amino acid changes in HA were viable, and some had altered antigenic properties. H5 HA can therefore accommodate many mutations in regions that affect antigenicity. The substantial plasticity of H5 HA may facilitate the emergence of novel antigenic variants.

Chronic low-grade inflammation can cause several metabolic syndromes. Patients with psoriasis, a chronic immunological skin inflammation, often develop diabetes. However, it is not clear to date how psoriasis leads to, or is correlated with, glucose intolerance. Here, we investigate whether psoriasis itself is correlated with hyperglycemia in humans and mice. In patients, the severity of psoriasis was correlated with high blood glucose levels, and treatment of psoriasis by phototherapy improved insulin secretion. Imiquimod-induced systemic and cutaneous inflammation in mice, with features of human psoriasis, also resulted in hyperglycemia. Although it should be determined if psoriasis-like cutaneous inflammation alone can induce hyperglycemia, imiquimod-treated mice showed impairment of insulin secretion without significant islet inflammation. Administration of anti-IL-17A monoclonal antibody improved hyperglycemia in patients with psoriasis and imiquimod-treated mice with psoriasiform features. These results suggest that hyperglycemia is highly associated with psoriasis, mainly through IL-17.