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Throughout the recent COVID-19 pandemic, South Korea led national efforts to develop vaccines and therapeutics for SARS-CoV-2. The project proceeded as follows: 1) evaluation system setup (including Animal Biosafety Level 3 (ABSL3) facility alliance, standardized nonclinical evaluation protocol, and laboratory information management system), 2) application (including committee review and selection), and 3) evaluation (including expert judgment and reporting). After receiving 101 applications, the selection committee reviewed pharmacokinetics, toxicity, and efficacy data and selected 32 final candidates. In the nonclinical efficacy test, we used golden Syrian hamsters and human angiotensin-converting enzyme 2 transgenic mice under a cytokeratin 18 promoter to evaluate mortality, clinical signs, body weight, viral titer, neutralizing antibody presence, and histopathology. These data indicated eight new drugs and one repositioned drug having significant efficacy for COVID-19. Three vaccine and four antiviral drugs exerted significant protective activities against SARS-CoV-2 pathogenesis. Additionally, two anti-inflammatory drugs showed therapeutic effects on lung lesions and weight loss through their mechanism of action but did not affect viral replication. Along with systematic verification of COVID-19 animal models through large-scale studies, our findings suggest that ABSL3 multicenter alliance and nonclinical evaluation protocol standardization can promote reliable efficacy testing against COVID-19, thus expediting medical product development.
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COVID-19 , Animales , Cricetinae , Ratones , Humanos , SARS-CoV-2 , Pandemias , Anticuerpos Neutralizantes , Mesocricetus , Modelos Animales de EnfermedadRESUMEN
Identification of highly selective type II kinase inhibitors is described. Two different chiral peptidomimetic scaffolds were introduced on the tail region of non-selective type II kinase inhibitor GNF-7 to enhance the selectivity. Kinome-wide selectivity profiling analysis showed that type II kinase inhibitor 7a potently inhibited Lck kinase with great selectivity (IC50 of 23.0 nM). It was found that 7a and its derivatives possessed high selectivity for Lck over even structurally conserved all Src family kinases. We also observed that 7a inhibited Lck activation in Jurkat T cells. Moreover, 7a was found to alleviate clinical symptoms in DSS-induced colitis mice. This study provides a novel insight into the design of selective type II kinase inhibitors by adopting chiral peptidomimetic moieties on the tail region.
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Peptidomiméticos , Animales , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito , Ratones , Peptidomiméticos/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Familia-src QuinasasRESUMEN
BACKGROUND: C1q has been reported to reveal complement-independent roles in immune and non-immune cells. C1q binds to its specific receptors to regulate distinct functions that rely on the environment and cell types. Discoidin domain receptor 2 (DDR2) is activated by collagen and functions in wound healing by controlling matrix metalloproteinase (MMP) expression. Since C1q exhibits a collagen-like structure, we hypothesized that C1q might engage DDR2 to regulate wound healing and extracellular matrix (ECM) remodeling. METHODS: Cell-based assay, proximity ligation assay, ELISA, and surface plasmon analysis were utilized to investigate DDR2 and C1q binding. We also investigate the C1q-mediated in vitro wound healing ability using the human fibrosarcoma cell line, HT1080. RESULTS: C1q induced the phosphorylation of DDR2, p38 kinase, and ERK1/2. C1q and DDR2 binding improved cell migration and induced MMP2 and MMP9 expression. DDR2-specific shRNA reduced C1q-mediated cell migration for wound healing. CONCLUSIONS: C1q is a new DDR2 ligand that promotes wound healing. These findings have therapeutic implications in wound healing-related diseases.
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Movimiento Celular/fisiología , Colágeno/metabolismo , Complemento C1q/metabolismo , Receptor con Dominio Discoidina 2/metabolismo , Secuencia de Aminoácidos , Línea Celular Tumoral , Colágeno/química , Complemento C1q/química , Receptor con Dominio Discoidina 1/genética , Receptor con Dominio Discoidina 1/metabolismo , Receptor con Dominio Discoidina 2/genética , Humanos , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , Microscopía Confocal , Péptidos/metabolismo , Fosforilación , Unión Proteica , Transducción de Señal , Cicatrización de Heridas/fisiologíaRESUMEN
High mobility group box-1 (HMGB1) is involved in various diseases and is associated with the resistance of many types of human cancers to chemotherapy; however, its role in cancer metastasis remains unexplored. This study examined the HMGB1 status of both highly and poorly metastatic cancer cells in response to genotoxic stress. The weakly and highly metastatic mouse melanoma cell lines (B16 vs. B16-F10), human melanoma cell lines (SK-MEL-28 vs. SK-MEL-24), colon cancer cell lines (DLD-1 vs. LS174T), and wild-type (WT) vs. HMGB1 knockout (KO) mouse embryonic fibroblasts (MEFs) were treated with doxorubicin (Dox) and camptothecin (CPT), and then cellular morphology, senescence-associated ß-galactosidase staining, lactate dehydrogenase release, and caspase-3 activation were used to assess cell fate. To investigate the role of HMGB1 in p21 expression, HMGB1 and p21 expressions were examined by Western blotting, and the HMGB1-mediated p21 promoter luciferase assay was performed after small interfering RNA or overexpression of HMGB1 prior to Dox treatment. Although highly metastatic mouse melanoma B16-F10 cells preferred senescence, with persistent HMGB1 expression, poorly metastatic B16 cells entered apoptosis, with decreasing HMGB1 levels via cleavage under Dox treatment. Similarly, more metastatic human melanoma SK-MEL-24 and human colon cancer LS174T cells underwent senescence, whereas fewer metastatic melanoma SK-MEL-28 and DLD-1 cells exhibited apoptosis under Dox stimulation. In senescent B16-F10, SK-MEL-24, and LS174T cells treated with Dox, p21 levels were increased by persistent HMGB1 expression. Furthermore, HMGB1 depletion caused a senescence-apoptosis shift with p21 down-regulation in B16-F10 cells, and HMGB1 overexpression switched from apoptosis to senescence concomitantly with increased p21 expression in B16 cells after Dox treatment. The same effects were observed in both cell pairs of mouse melanoma and human colon cancer cells treated with CPT, another genotoxic stressor. Indeed, although WT MEF entered senescence accompanied by p21 increase, HMGB1 KO underwent apoptosis with p21 decrease by Dox treatment. In our cell model system, we demonstrated that highly metastatic cancer cells preferentially enter senescence, whereas apoptosis predominates in weakly metastatic cancer cells under genotoxic stress, which depends on the presence or absence of HMGB1, suggesting that the HMGB1-p21 axis is required for genotoxic stress-induced senescence. These findings suggest that HMGB1 modulation of cancers with different metastatic status could be a strategy for selectively enforcing tumor suppression.-Lee, J.-J., Park, I. H., Rhee, W. J., Kim, H. S., Shin, J.-S. HMGB1 modulates the balance between senescence and apoptosis in response to genotoxic stress.
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Apoptosis , Senescencia Celular , Daño del ADN , Proteína HMGB1/metabolismo , Animales , Camptotecina/toxicidad , Línea Celular Tumoral , Células Cultivadas , Neoplasias del Colon/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Doxorrubicina/toxicidad , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Proteína HMGB1/genética , Humanos , Melanoma/metabolismo , RatonesRESUMEN
BACKGROUND: Cancer stem cells (CSCs), a subpopulation in tumors, are known to cause drug resistance, tumor recurrence and metastasis. Based on the characteristic formation of mammospheres in in vitro conditions, the mammosphere formation assay has become an essential tool for quantifying CSC activity in breast cancer research. However, manual counting of mammospheres is a time-consuming process that is not amenable to high-throughput screening, and there are occasional inaccuracies in the process of determining the mammosphere diameter. In this study, we proposed a novel automated counting method of mammosphere using the National Institute of Standards and Technology (NIST)'s Integrated Colony Enumerator (NICE) with a screening of protein kinase library. METHODS: Human breast cancer cell line MCF-7 was used for evaluation of tumor sphere efficiency, migration, and phenotype transition. Cell viability was assessed using MTT assay, and CSCs were identified by an analysis of CD44 expression and ALDEFLUOR assay using flow cytometry. Automated counting of mammosphere using NICE program was performed with a comparison to the result of manual counting. After identification of inhibitors to ameliorate CSC formation by screening a library of 79 protein kinase inhibitors using automated counting in primary, secondary and tertiary mammosphere assay, the effect of selected kinase inhibitors on migration, colony formation and epithelial-to-mesenchymal transition (EMT) of MCF-7 cells was investigated. RESULTS: Automated counting of mammosphere using NICE program was an easy and less time-consuming process (<1 min for reading 6-well plate) which provided a comparable result with manual counting. Inhibition of calcium/calmodulin-dependent protein kinase II (CaMKII), Janus kinase-3 (JAK-3), and IκB kinase (IKK) were identified to decrease the formation of MCF-7-derived CSCs in primary, secondary and tertiary mammosphere assay. These protein kinase inhibitors alleviated TGF-ß1-induced migration, colony formation and EMT of MCF-7 cells. CONCLUSIONS: We have developed a novel automated cell-based screening method which provided an easy, accurate and reproducible way for mammosphere quantification. This study is the first to show the efficacy of an automated medium-throughput mammosphere-counting method in CSC-related research with an identification of protein kinase inhibitors to ameliorate CSC formation.
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BACKGROUND: Alopecic and aseptic nodule of the scalp (AANS) is a rare disease entity first reported in 1992 as pseudocyst of the scalp (PCS). Controversy exists regarding the histopathology and etiology of reported cases. OBJECTIVE: We performed this study to analyze the clinical and histopathologic features of AANS/PCS in Korean patients. METHODS: A retrospective review of medical records from 2008 to 2013 at Inje University Busan Paik Hospital was performed. RESULTS: Eleven patients were enrolled. All patients were male, and their mean age was 21.6 years. Most patients had a solitary nodule (10/11) located predominantly on the vertex. The mean nodule size was 20 mm. Inflammatory cell infiltration in the deep dermis was a histologic feature of AANS/PCS. Eight patients showed granulomatous infiltration. All patients were treated with short-term antibiotics and intralesional steroid injection. CONCLUSION: Our results suggest that dermatologists should consider AANS when diagnosing an alopecic nodule on the scalp.
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Alopecia/complicaciones , Alopecia/patología , Quiste Epidérmico/complicaciones , Quiste Epidérmico/patología , Dermatosis del Cuero Cabelludo/etiología , Dermatosis del Cuero Cabelludo/patología , Adolescente , Adulto , Alopecia/tratamiento farmacológico , Combinación Amoxicilina-Clavulanato de Potasio/uso terapéutico , Antibacterianos/uso terapéutico , Antiinflamatorios/administración & dosificación , Cefalosporinas/uso terapéutico , Niño , Quimioterapia Combinada , Quiste Epidérmico/tratamiento farmacológico , Humanos , Inyecciones Intralesiones , Masculino , República de Corea , Estudios Retrospectivos , Dermatosis del Cuero Cabelludo/tratamiento farmacológico , Triamcinolona/administración & dosificación , Adulto JovenRESUMEN
Although it is well known that pneumococcal conjugate vaccines provide cross-protection against some vaccine-related serotypes, these mechanisms are still unclear. This study was performed to investigate the role of cross-protective IgM antibodies against vaccine-related serotypes 6A, 6C, and 19A induced in children aged 12-23 months after immunization with 7-valent pneumococcal conjugate vaccine (PCV7). We obtained serum samples from 18 Korean children aged 12-23 months after a PCV7 booster immunization. The serum IgG and IgM concentrations of serotypes 6B and 19F were measured by enzyme-linked immunosorbent assay (ELISA) in serum. The opsonic indices (OIs) against vaccine serotypes 6B and 19F and vaccine-related serotypes 6A, 6C, and 19A were determined by an opsonophagocytic killing assay (OPA) in IgM-depleted and control serum. Both IgG and IgM antibodies in ELISA and opsonic indices in OPA against serotypes 6B and 19F were demonstrated in the immune serum. IgM depletion decreased the OIs against vaccine serotypes 6B (geometric means of OIs (GMIs) of 3,009 vs. 1,396, 38% reduction) and 19F (1,117 vs. 750, 36% reduction). In addition, IgM depletion markedly decreased the OIs against vaccine-related serotypes 6A (GMIs of 961 vs. 329, 70% reduction), 6C (432 vs. 185, 72% reduction), and 19A (301 vs. 166, 58% reduction). The booster immunization PCV7 induced protective antibodies in the form of both IgG and IgM isotypes. IgM antibodies contributed to eliciting cross-protection against vaccine-related serotypes as well as against vaccine serotypes.
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Vacuna Neumocócica Conjugada Heptavalente/inmunología , Inmunoglobulina M/sangre , Infecciones Neumocócicas/prevención & control , Vacunas Neumococicas/inmunología , Anticuerpos Antibacterianos/sangre , Anticuerpos Neutralizantes/sangre , Ensayo de Inmunoadsorción Enzimática , Humanos , Lactante , Serogrupo , Streptococcus pneumoniae/inmunologíaRESUMEN
Recently, it has been reported that some non-typable (NT) Streptococcus pneumoniae isolates from Korea and other countries contained a novel gene pspK in the capsular polysaccharide synthesis (cps) locus. In this study, we investigated the presence of pspK in 120 NT S. pneumoniae isolates from 12 Asian countries; isolate characteristics were also examined. The presence of pspK was assayed by PCR. Clonality of NT S. pneumoniae isolates containing pspK was investigated by MLST and PFGE. Antimicrobial susceptibility testing was performed and the structure of pspK was also determined. Nineteen NT isolates (15.8â%) were identified as containing pspK: two isolates from Korea, four from Vietnam, two from Hong Kong, eight from Thailand, and one each from Taiwan, the Philippines and Saudi Arabia. Seven isolates from Korea, Vietnam and Thailand were identified as ST1106, whereas just one or two belonged to ST310, ST393, ST10137, ST2754 or ST4136. All but one of the ST1106 NT isolates showed non-susceptibility to penicillin, and all isolates were resistant to cefuroxime, erythromycin, clindamycin and trimethoprim/sulfamethoxazole. The structure of pspK was similar amongst 20 isolates, which had a R1-R2-like region and a variable number of repeats in the repetitive region. However, one isolate (P05-11) from the Philippines lacked the R1-R2 region. NT S. pneumoniae isolates containing pspK were distributed across several Asian countries. Although MLST analysis suggested that most pspK-containing NT S. pneumoniae isolates may have emerged independently, ST1106 isolates with the selective advantage of antimicrobial resistance may have disseminated clonally throughout the countries.
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Proteínas de la Membrana Bacteriana Externa/genética , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/clasificación , Streptococcus pneumoniae/genética , Antibacterianos/farmacología , Asia/epidemiología , Farmacorresistencia Bacteriana/genética , Genotipo , Geografía , Humanos , Pruebas de Sensibilidad Microbiana , Infecciones Neumocócicas/epidemiología , Prevalencia , Streptococcus pneumoniae/aislamiento & purificaciónRESUMEN
The prevalence of serogroup 6 among 1,206 Streptococcus pneumoniae clinical isolates collected from Korean hospitals over three periods (1996 to 2001, 2004 to 2006, and 2008 to 2009) was investigated. The number of serogroup 6 isolates increased from 9.7 to 17.5% over the three periods. While the proportion of serotype 6A and 6D isolates increased significantly, that of serotype 6B isolates decreased. Twenty-four isolates (2.0%) were typed as the recently identified putative serotype 6E or genetic variants of serotype 6B. The results suggest that the lack of change in frequency of serotype 6B, in spite of the introduction of the PCV7 vaccine as seen in previous studies in South Korea, might be due mainly to the improper inclusion of putative serotype 6E in serotype 6B. All but three serotype 6E isolates belonged to CC90, indicating their clonal expansion.
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Infecciones Neumocócicas/epidemiología , Infecciones Neumocócicas/microbiología , Streptococcus pneumoniae/clasificación , Streptococcus pneumoniae/aislamiento & purificación , Antibacterianos/farmacología , Variación Genética , Genotipo , Hospitales , Humanos , Pruebas de Sensibilidad Microbiana , Tipificación de Secuencias Multilocus , Prevalencia , República de Corea/epidemiología , Serogrupo , Streptococcus pneumoniae/efectos de los fármacos , Streptococcus pneumoniae/genéticaRESUMEN
Intranasal infection is commonly used to establish a SARS-CoV-2 mouse model due to its non-invasive procedures and a minimal effect from the operation itself. However, mice intranasally infected with SARS-CoV-2 have a high mortality rate, which limits the utility of this model for exploring therapeutic strategies and the sequelae of non-fatal COVID-19 cases. To resolve these limitations, an aerosolised viral administration method has been suggested. However, an in-depth pathological analysis comparing the two models is lacking. Here, we show that inhalation and intranasal SARS-CoV-2 (106 PFU) infection models established in K18-hACE2 mice develop unique pathological features in both the respiratory and central nervous systems, which could be directly attributed to the infection method. While the inhalation-infection model exhibited relatively milder pathological parameters, it closely mimicked the prevalent chest CT pattern observed in COVID-19 patients with focal, peripheral lesions and fibrotic scarring in the recuperating lung. We also found the evidence of direct neuron-invasion from the olfactory receptor neurons to the olfactory bulb in the intranasal model and showed the trigeminal nerve as an alternative route of transmission to the brain in inhalation infected mice. Even after viral clearance confirmed at 14 days post-infection, mild lesions were still found in the brain of inhalation-infected mice. These findings suggest that the inhalation-infection model has advantages over the intranasal-infection model in closely mimicking the pathological features of non-fatal symptoms of COVID-19, demonstrating its potential to study the sequelae and possible interventions for long COVID.
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COVID-19 , Modelos Animales de Enfermedad , Pulmón , SARS-CoV-2 , Animales , COVID-19/patología , COVID-19/virología , Ratones , Pulmón/patología , Pulmón/virología , Enzima Convertidora de Angiotensina 2/metabolismo , Enzima Convertidora de Angiotensina 2/genética , Bulbo Olfatorio/patología , Bulbo Olfatorio/virología , Humanos , Administración Intranasal , Femenino , Neuronas Receptoras Olfatorias/virología , Neuronas Receptoras Olfatorias/metabolismoRESUMEN
Viral load and the duration of viral shedding of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) are important determinants of the transmission of coronavirus disease 2019. In this study, we examined the effects of viral doses on the lung and spleen of K18-hACE2 transgenic mice by temporal histological and transcriptional analyses. Approximately, 1×105 plaque-forming units (PFU) of SARS-CoV-2 induced strong host responses in the lungs from 2 days post inoculation (dpi) which did not recover until the mice died, whereas responses to the virus were obvious at 5 days, recovering to the basal state by 14 dpi at 1×102 PFU. Further, flow cytometry showed that number of CD8+ T cells continuously increased in 1×102 PFU-virus-infected lungs from 2 dpi, but not in 1×105 PFU-virus-infected lungs. In spleens, responses to the virus were prominent from 2 dpi, and number of B cells was significantly decreased at 1×105 PFU; however, 1×102 PFU of virus induced very weak responses from 2 dpi which recovered by 10 dpi. Although the defense responses returned to normal and the mice survived, lung histology showed evidence of fibrosis, suggesting sequelae of SARS-CoV-2 infection. Our findings indicate that specific effectors of the immune response in the lung and spleen were either increased or depleted in response to doses of SARS-CoV-2. This study demonstrated that the response of local and systemic immune effectors to a viral infection varies with viral dose, which either exacerbates the severity of the infection or accelerates its elimination.
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BACKGROUND: The global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has led to approximately 500 million cases and 6 million deaths worldwide. Previous investigations into the pathophysiology of SARS-CoV-2 primarily focused on peripheral blood mononuclear cells from patients, lacking detailed mechanistic insights into the virus's impact on inflamed tissue. Existing animal models, such as hamster and ferret, do not faithfully replicate the severe SARS-CoV-2 infection seen in patients, underscoring the need for more relevant animal system-based research. METHODS: In this study, we employed single-cell RNA sequencing (scRNA-seq) with lung tissues from K18-hACE2 transgenic (TG) mice during SARS-CoV-2 infection. This approach allowed for a comprehensive examination of the molecular and cellular responses to the virus in lung tissue. FINDINGS: Upon SARS-CoV-2 infection, K18-hACE2 TG mice exhibited severe lung pathologies, including acute pneumonia, alveolar collapse, and immune cell infiltration. Through scRNA-seq, we identified 36 different types of cells dynamically orchestrating SARS-CoV-2-induced pathologies. Notably, SPP1+ macrophages in the myeloid compartment emerged as key drivers of severe lung inflammation and fibrosis in K18-hACE2 TG mice. Dynamic receptor-ligand interactions, involving various cell types such as immunological and bronchial cells, defined an enhanced TGFß signaling pathway linked to delayed tissue regeneration, severe lung injury, and fibrotic processes. INTERPRETATION: Our study provides a comprehensive understanding of SARS-CoV-2 pathogenesis in lung tissue, surpassing previous limitations in investigating inflamed tissues. The identified SPP1+ macrophages and the dysregulated TGFß signaling pathway offer potential targets for therapeutic intervention. Insights from this research may contribute to the development of innovative diagnostics and therapies for COVID-19. FUNDING: This research was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MSIT) (2020M3A9I2109027, 2021R1A2C2004501).
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COVID-19 , Melfalán , gammaglobulinas , Animales , Cricetinae , Ratones , Humanos , SARS-CoV-2 , Leucocitos Mononucleares , Hurones , Bronquios , Factor de Crecimiento Transformador beta , Ratones Transgénicos , Modelos Animales de Enfermedad , PulmónRESUMEN
The coronavirus pandemic has accelerated the development of next-generation vaccination technology to combat future pandemic outbreaks. Mucosal vaccination effectively protects the mucosal surfaces, the primary sites of viral entry, by inducing the secretion of immunoglobulin A (IgA) and humoral IgG. Here, a dissolving microneedle (DMN) is adopted as a mucosal vaccine delivery platform to directly penetrate the sublingual site, which is rich in antigen-presenting cells (APCs) and lymphoid tissues. The sublingual dissolving microneedle (SLDMN) vaccination platform comprised a micropillar-based compartment and a 3D-printed SLDMN applicator as a substitute for the DMN patch. The penetration efficacy of SLDMNs is assessed using in vitro optical coherence tomography (OCT) and in vivo histological analysis. The efficacy of SLDMN is also evaluated in a vaccine form using the recombinant spike (S1) protein of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Furthermore, SLDMN is used to challenge transgenic mice expressing human angiotensin-converting enzyme 2 (hACE2) receptors. Its effects are evaluated on antibody production, survival rate, and inflammation attenuation after infection compared to the intramuscular (IM) injections. Overall, SLDMN effectively induced mucosal immunity via IgA secretion, attenuated lung inflammation, and lowered the levels of cytokines and chemokines, which may prevent the "cytokine storm" after SARS-CoV-2 infection.
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COVID-19 , Vacunas Virales , Ratones , Animales , Humanos , SARS-CoV-2 , Anticuerpos Antivirales , Inmunidad Mucosa , COVID-19/prevención & control , Inmunoglobulina A/análisisRESUMEN
The integration of artificial intelligence (AI) into drug discovery has markedly advanced the search for effective therapeutics. In our study, we employed a comprehensive computational-experimental approach to identify potential anti-SARS-CoV-2 compounds. We developed a predictive model to assess the activities of compounds based on their structural features. This model screened a library of approximately 700,000 compounds, culminating in the selection of the top 100 candidates for experimental validation. In vitro assays on human intestinal epithelial cells (Caco-2) revealed that 19 of these compounds exhibited inhibitory activity. Notably, eight compounds demonstrated dose-dependent activity in Vero cell lines, with half-maximal effective concentration (EC50) values ranging from 1 µM to 7 µM. Furthermore, we utilized a clustering approach to pinpoint potential nucleoside analog inhibitors, leading to the discovery of two promising candidates: azathioprine and its metabolite, thioinosinic acid. Both compounds showed in vitro activity against SARS-CoV-2, with thioinosinic acid also significantly reducing viral loads in mouse lungs. These findings underscore the utility of AI in accelerating drug discovery processes.
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Evidence suggests that the human respiratory tract, as with the gastrointestinal tract, has evolved to its current state in association with commensal microbes. However, little is known about how the airway microbiome affects the development of airway immune system. Here, we uncover a previously unidentified mode of interaction between host airway immunity and a unique strain (AIT01) of Staphylococcus epidermidis, a predominant species of the nasal microbiome. Intranasal administration of AIT01 increased the population of neutrophils and monocytes in mouse lungs. The recruitment of these immune cells resulted in the protection of the murine host against infection by Pseudomonas aeruginosa, a pathogenic bacterium. Interestingly, an AIT01-secreted protein identified as GAPDH, a well-known bacterial moonlighting protein, mediated this protective effect. Intranasal delivery of the purified GAPDH conferred significant resistance against other Gram-negative pathogens (Klebsiella pneumoniae and Acinetobacter baumannii) and influenza A virus. Our findings demonstrate the potential of a native nasal microbe and its secretory protein to enhance innate immune defense against airway infections. These results offer a promising preventive measure, particularly relevant in the context of global pandemics.
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection induces excessive pro-inflammatory cytokine release and cell death, leading to organ damage and mortality. High-mobility group box 1 (HMGB1) is one of the damage-associated molecular patterns that can be secreted by pro-inflammatory stimuli, including viral infections, and its excessive secretion levels are related to a variety of inflammatory diseases. Here, the aim of the study was to show that SARS-CoV-2 infection induced HMGB1 secretion via active and passive release. Active HMGB1 secretion was mediated by post-translational modifications, such as acetylation, phosphorylation, and oxidation in HEK293E/ACE2-C-GFP and Calu-3 cells during SARS-CoV-2 infection. Passive release of HMGB1 has been linked to various types of cell death; however, we demonstrated for the first time that PANoptosis, which integrates other cell death pathways, including pyroptosis, apoptosis, and necroptosis, is related to passive HMGB1 release during SARS-CoV-2 infection. In addition, cytoplasmic translocation and extracellular secretion or release of HMGB1 were confirmed via immunohistochemistry and immunofluorescence in the lung tissues of humans and angiotensin-converting enzyme 2-overexpressing mice infected with SARS-CoV-2.
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Coronavirus Disease 2019 (COVID-19) pandemic is severely impacting the world, and tremendous efforts have been made to deal with it. Despite many advances in vaccines and therapeutics, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants remains an intractable challenge. We present a bivalent Receptor Binding Domain (RBD)-specific synthetic antibody, specific for the RBD of wild-type (lineage A), developed from a non-antibody protein scaffold composed of LRR (Leucine-rich repeat) modules through phage display. We further reinforced the unique feature of the synthetic antibody by constructing a tandem dimeric form. The resulting bivalent form showed a broader neutralizing activity against the variants. The in vivo neutralizing efficacy of the bivalent synthetic antibody was confirmed using a human ACE2-expressing mouse model that significantly alleviated viral titer and lung infection. The present approach can be used to develop a synthetic antibody showing a broader neutralizing activity against a multitude of SARS-CoV-2 variants.
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COVID-19 , SARS-CoV-2 , Animales , Ratones , Humanos , SARS-CoV-2/genética , Anticuerpos , Técnicas de Visualización de Superficie Celular , Glicoproteína de la Espiga del Coronavirus/genética , Anticuerpos Neutralizantes/uso terapéutico , Anticuerpos Antivirales/uso terapéuticoRESUMEN
BACKGROUND: As the number of large-scale studies involving multiple organizations producing data has steadily increased, an integrated system for a common interoperable format is needed. In response to the coronavirus disease 2019 (COVID-19) pandemic, a number of global efforts are underway to develop vaccines and therapeutics. We are therefore observing an explosion in the proliferation of COVID-19 data, and interoperability is highly requested in multiple institutions participating simultaneously in COVID-19 pandemic research. RESULTS: In this study, a laboratory information management system (LIMS) approach has been adopted to systemically manage various COVID-19 non-clinical trial data, including mortality, clinical signs, body weight, body temperature, organ weights, viral titer (viral replication and viral RNA), and multiorgan histopathology, from multiple institutions based on a web interface. The main aim of the implemented system is to integrate, standardize, and organize data collected from laboratories in multiple institutes for COVID-19 non-clinical efficacy testings. Six animal biosafety level 3 institutions proved the feasibility of our system. Substantial benefits were shown by maximizing collaborative high-quality non-clinical research. CONCLUSIONS: This LIMS platform can be used for future outbreaks, leading to accelerated medical product development through the systematic management of extensive data from non-clinical animal studies.
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[This corrects the article DOI: 10.3389/fimmu.2022.1055811.].
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Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causing coronavirus disease 2019 (COVID-19) has been a global health concern since 2019. The viral spike protein infects the host by binding to angiotensin-converting enzyme 2 (ACE2) expressed on the cell surface, which is then processed by type II transmembrane serine protease. However, ACE2 does not react to SARS-CoV-2 in inbred wild-type mice, which poses a challenge for preclinical research with animal models, necessitating a human ACE2 (hACE2)-expressing transgenic mouse model. Cytokeratin 18 (K18) promoter-derived hACE2 transgenic mice [B6.Cg-Tg(K18-ACE2)2Prlmn/J] are widely used for research on SARS-CoV-1, MERS-CoV, and SARS-CoV-2. However, SARS-CoV-2 infection is lethal at ≥105 PFU and SARS-CoV-2 target cells are limited to type-1 alveolar pneumocytes in K18-hACE2 mice, making this model incompatible with infections in the human lung. Hence, we developed lung-specific SARS-CoV-2 infection mouse models with surfactant protein B (SFTPB) and secretoglobin family 1a member 1 (Scgb1a1) promoters. After inoculation of 105 PFU of SARS-CoV-2 to the K18-hACE2, SFTPB-hACE2, and SCGB1A1-hACE2 models, the peak viral titer was detected at 2 days post-infection and then gradually decreased. In K18-hACE2 mice, the body temperature decreased by approximately 10°C, body weight decreased by over 20%, and the survival rate was reduced. However, SFTPB-hACE2 and SCGB1A1-hACE2 mice showed minimal clinical signs after infection. The virus targeted type I pneumocytes in K18-hACE2 mice; type II pneumocytes in SFTPB-hACE2 mice; and club, goblet, and ciliated cells in SCGB1A1-hACE2 mice. A time-dependent increase in severe lung lesions was detected in K18-hACE2 mice, whereas mild lesions developed in SFTPB-hACE2 and SCGB1A1-hACE2 mice. Spleen, small intestine, and brain lesions developed in K18-hACE2 mice but not in SFTPB-hACE2 and SCGB1A1-hACE2 mice. These newly developed SFTPB-hACE2 and SCGB1A1-hACE2 mice should prove useful to expand research on hACE2-mediated respiratory viruses.