Establishment of multicenter COVID-19 therapeutics preclinical test system in Republic of Korea.

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.

Dermoscopic findings and histopathological correlation in large cell acanthoma.

Since large cell acanthoma (LCA) has many overlapping clinical and histopathological features with other epidermal pigmented tumours, an additional method to differentiate it would be of great clinical significance. A retrospective study was performed on 33 lesions (26 patients) to identify distinct dermoscopic findings of LCA and to describe dermoscopic-histopathological correlations. The results revealed that dermoscopy significantly aids in the distinction of LCA from other epidermal tumours included in the differential diagnosis. Yellow opaque homogeneous background, brown dots, and moth-eaten border are common findings, and prominent skin markings and short white streaks are additional distinguishing features. Several important findings that are common in other diseases are rare in LCA.

Human papillomavirus genotype distribution of genital keratotic lesions.

Genital keratotic lesions include bowenoid papulosis (BP), which histologically resembles squamous cell carcinoma in situ containing high-risk HPV, condyloma acuminatum (CA) that is a genital wart containing mostly low-risk HPV, and genital seborrheic keratosis (GSK), which is a benign epidermal tumour lacking a clear etiologic relationship with HPV. This study compared HPV genotype distributions among BP, CA and GSK and revealed that BP and GSK were related to high-risk HPV whereas CA was related to low-risk HPV. It is plausible that GSK is a distinct epidermal tumour often related to high-risk HPV rather than merely a senescent form of CA considering the overall discrepancy in the frequency distribution of HPV genotypes along with histopathological differences, and the detection of high-risk HPV in GSK alerts physicians to consider more active treatment and continued follow-ups.

PTK7, a Catalytically Inactive Receptor Tyrosine Kinase, Increases Oncogenic Phenotypes in Xenograft Tumors of Esophageal Squamous Cell Carcinoma KYSE-30 Cells.

Protein tyrosine kinase 7 (PTK7), a catalytically defective receptor protein tyrosine kinase, is upregulated in tumor tissues and cell lines of esophageal squamous cell carcinoma (ESCC). We showed that PTK7 plays an oncogenic role in various ESCC cell lines. However, its role as an oncogene has not been demonstrated in vivo. Here, we examined the influence of PTK7 on the tumorigenic potential of ESCC KYSE-30 cells, which are known to establish xenograft tumors. Overexpression of PTK7 enhanced the proliferation, adhesion, wound healing, and migration of KYSE-30 cells, and these effects were reversed by the knockdown of PTK7. PTK7 overexpression and knockdown, respectively, increased and decreased the tyrosine phosphorylation of cellular proteins and the phosphorylation of ERK, AKT, and FAK, which are important for cell proliferation, survival, adhesion, and migration. Additionally, PTK7 overexpression and silencing, respectively, increased and decreased the weight, volume, and number of Ki-67-positive proliferating cells in xenograft tumors of KYSE-30 cells. Therefore, we propose that PTK7 plays an important role in the tumorigenesis of ESCC cells in vivo and is a potential therapeutic target for ESCC.

Low-dose Methotrexate Treatment for Solitary or Localized Primary Cutaneous Anaplastic Large Cell Lymphoma: A Long-term Follow-up Study.

Although low-dose methotrexate (MTX) has been used widely in treatment of a variety of dermatological diseases, including multifocal primary cutaneous anaplastic large cell lymphoma (PCALCL), it has not been established for use in the treatment guidelines for solitary or localized PCALCL. Furthermore, there has been no report of long-term follow-up data in Asian patients with PCALCL treated with low-dose MTX. To investigate the effectiveness and clinical outcome of treatment with low-dose MTX, clinical and long-term follow-up data of 7 patients with solitary or localized PCALCL were analysed retrospectively. Of the 7 patients, 6 (85.7%) showed a complete response and 1 (14.3%) showed partial remission. During follow-up, mean duration of 92.1 months, 5 patients developed one or more cutaneous relapses. At the last follow-up, all of the patients with PCALCL were alive without disease. These results indicate that low-dose MTX is a highly effective and safe treatment for solitary or localized PCALCL as well as multiple relapsed lesions.

The effectiveness of high-dose ultraviolet A-1 phototherapy for acute exacerbation of atopic dermatitis in Asians.

BACKGROUND/PURPOSE: Atopic dermatitis (AD) is an inflammatory skin disease characterized by a chronic course of exacerbations and remissions. High-dose ultraviolet A-1 (UVA-1) phototherapy has been effective in the treatment of acute exacerbations of AD. However, there have been no case studies in Asian patients to date. We investigated the effectiveness of high-dose UVA-1 phototherapy for treating acute exacerbation of AD in Asian patients. METHOD: This study included 16 patients with acute exacerbation of AD. High-dose (100 J/cm2 ) regimens of UVA-1 therapy were employed. Therapeutic effectiveness was assessed based on the findings of clinical examinations and scoring of AD (SCORAD) index before treatment and after the 5th and 10th sessions of treatment. Additionally, side effects and recurrence during follow-up were retrospectively evaluated. RESULTS: The patients were between 7 and 50 years of age, with a mean age of 25.8 years. The SCORAD index was between 41 and 89.5, with a mean score of 64.9. Among the 16 patients, two patients discontinued treatment due to the aggravation of erythema and pruritus. Of the 14 patients who completed the 10 sessions of high-dose UVA-1 phototherapy, nine patients (64.3%) showed complete remission and five patients (35.7%) showed partial remission. The mean SCORAD index reduced from 64.9 (before treatment) to 23.3 (after the 10th session of treatment). CONCLUSION: This is the first case study of high-dose UVA-1 phototherapy for acute exacerbation of AD in Asian patients, suggesting that high-dose UVA-1 phototherapy can be a well-tolerated and effective treatment for acute exacerbated AD. Future large-scale prospective studies are needed.

Burden of male hardcore smokers and its characteristics among those eligible for lung cancer screening.

BACKGROUND: There are few data available about hardcore smokers and their behavioral characteristics among the lung cancer screening (LCS) population. The study investigated the burden of hardcore smokers within the LCS population, and determine the characteristics of hardcore smokers using nationally representative data in South Korea. METHODS: We used data from 2007 to 2012 from the Korean National Health and Nutrition Examination Survey. This study enrolled current male smokers aged 55-74 years. Among them, subjects eligible for LCS were defined as these populations with smoking histories of at least 30 PY. Hardcore smoking was defined as smoking >15 cigarettes per day, with no plan to quit, and having made no attempt to quit. Multivariate logistic regression analyses were used to estimate associations between hardcore smokers and various sociodemographic and other variables. RESULTS: The proportion of hardcore smokers among those who met LCS eligibility criteria decreased from 2007 to 2012 (from 39.07 to 29.47% of the population) but did not change significantly thereafter (P = 0.2770), and that proportion was consistently 10-15% higher than that of hardcore smokers among all male current smokers. The proportion without any plan to quit smoking decreased significantly from 54.35% in 2007 to 38.31% in 2012. However, the smokers who had made no intentional quit attempt in the prior year accounted for more than half of those eligible for LCS, and the proportion of such smokers did not change significantly during the study period (50.83% in 2007 and 51.03% in 2012). Multivariate logistic regression analyses showed that hardcore smokers were older (OR = 1.05, 95% confidence interval [CI] 1.01-1.09) than non-hardcore smokers. Hardcore smokers exhibited higher proportion of depression (OR = 6.55, 95% CI 1.75-24.61) and experienced extreme stress more frequently (OR = 1.93, 95% CI 1.13-3.29). Smokers who did not receive smoking cessation education within the past year were significantly more likely to be hardcore smokers (OR = 4.15, 95% CI 1.30-13.22). CONCLUSIONS: It is important to identify a subset of smokers unwilling or minimally motivated to quit within the context of lung cancer screening. Anti-smoking education should be enhanced to influence hardcore smokers' behavior.

The Multifaceted Role of Epithelial Membrane Protein 2 in Cancer: from Biomarker to Therapeutic Target.

Tetraspanin superfamily proteins not only facilitate the trafficking of specific proteins to distinct plasma membrane domains but also influence cell-to-cell and cell-extracellular matrix interactions. Among these proteins, Epithelial Membrane Protein 2 (EMP2), a member of the growth arrest-specific gene 3/peripheral myelin protein 22 (GAS3/PMP22) family, is known to affect key cellular processes. Recent studies have revealed that EMP2 modulates critical signaling pathways and interacts with adhesion molecules and growth factor receptors, underscoring its potential as a biomarker for cancer diagnosis and prognosis. These findings suggest that EMP2 expression patterns could provide valuable insights into tumorigenesis and metastasis. Moreover, EMP2 has emerged as a promising therapeutic target, with approaches aimed at inhibiting or modulating its activity showing potential to disrupt tumor growth and metastasis. This review provides a comprehensive overview of recent advances in understanding the multifaceted roles of EMP2 in cancer, with a focus on its underlying mechanisms and clinical significance.

NR1D1 deficiency in the tumor microenvironment promotes lung tumor development by activating the NLRP3 inflammasome.

Nuclear receptor Rev-erbα (NR1D1) is a major negative regulator of the circadian clock. Numerous studies have investigated the role of circadian clock-related factors in the tumorigenesis of multiple cancer types, but little is known about the role of NR1D1 in cancer development. In this study, we identified the role of NR1D1 in lung tumorigenesis using genetically engineered mouse models of Nr1d1. Although NR1D1 overexpression or knockdown had little effect on the proliferation of NSCLC cells in vitro, NR1D1 deficiency in the tumor microenvironment increased lung cancer development compared with the control in the orthotopic model. NR1D1-deficient mice showed increased NACHT, LRR, and PYD domain-containing protein 3 (NLRP3) inflammasome activation, and conditioned medium (CM) from NR1D1-deficient macrophages increased the proliferation and epithelial-mesenchymal transition (EMT) of lung cancer cells. Treatment with MCC950, a specific inhibitor of NLRP3 inflammasome, blocked tumorigenesis in NR1D1-deficient mice in an orthotopic lung cancer model. In addition, MCC950 treatment blocked the increased proliferation and EMT of cancer cells induced by CM from NR1D1-deficient macrophages in vitro. Our results showed that NR1D1 in the tumor microenvironment functions as a tumor suppressor by negatively regulating the NLRP3 inflammasome, suggesting that the NLRP3 inflammasome blockade via NR1D1 activation could be a therapeutic strategy to overcome lung cancer.

Chondroprotective Effects of Ulva prolifera on Osteoarthritis through MAPKs Signaling Inhibition.

Osteoarthritis (OA) is a typical degenerative disease that mainly appears in the elderly aged 65 and over. OA is characterized by inflammation and decomposition of the cartilage matrix due to irreversible wear and tear. Ulva prolifera, a green macroalgae species, contains polysaccharides, amino acids, polyunsaturated fatty acids, and polyphenols, which are major active components responsible for anti-inflammatory and antioxidant effects. This study evaluated the chondro-protective effect of 30% prethanol extract of U. prolifera (30% PeUP). Rat primary chondrocytes were pre-treated with 30% PeUP for 1 h before interleukin-1ß (10 ng/mL) stimulation. The production of nitrite, prostaglandin E2 (PGE2), collagen type II (Col II), and aggrecan (ACAN) were detected by Griess reagent and enzyme-linked immunosorbent assay. The protein expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase (COX)-2, matrix metalloproteinase (MMP)-1, MMP-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin (ADAMTS)-4, ADAMTS-5, and mitogen-activated protein kinases (MAPKs) (extracellular signal-regulated kinase 1/2, c-Jun N-terminal kinase, and p38) were assessed by western blot. Thirty percent of PeUP significantly inhibited the expression of nitrite, iNOS, PGE2, COX-2, MMP-1, MMP-3, MMP-13, ADMATS-4, and ADMATS-5 in interleukin (IL)-1ß-stimulated chondrocytes. Moreover, 30% PeUP decreased the IL-1ß-induced degradation of Col II and ACAN. Additionally, 30% of PeUP suppressed IL-1ß-induced phosphorylation of MAPKs. Therefore, 30% PeUP is a potential therapeutic agent to mitigate OA progression.

A Rapid and Noninvasive Method That Extracts Polymerase Chain Reaction-Ready Genomic DNA from Adult Zebrafish.

Genotyping usually entails analysis of the products of polymerase chain reaction (PCR) carried out with genomic DNA (gDNA) as template, and is employed for validation of mutant or transgenic organisms. For genotyping of adult zebrafish, gDNA is often extracted from clipped caudal fin or skin mucus through either alkaline lysis using NaOH or proteinase K (PK) treatment. Further purification of the gDNA using ethanol precipitation was optional. To develop a rapid and noninvasive method that extracts PCR-ready gDNA from adult zebrafish, we combined skin swabbing with PK treatment and demonstrated its efficiency. This method could be applied to a wide range of fish.

PRR16/Largen Induces Epithelial-Mesenchymal Transition through the Interaction with ABI2 Leading to the Activation of ABL1 Kinase.

Advanced or metastatic breast cancer affects multiple organs and is a leading cause of cancer-related death. Cancer metastasis is associated with epithelial-mesenchymal metastasis (EMT). However, the specific signals that induce and regulate EMT in carcinoma cells remain unclear. PRR16/Largen is a cell size regulator that is independent of mTOR and Hippo signalling pathways. However, little is known about the role PRR16 plays in the EMT process. We found that the expression of PRR16 was increased in mesenchymal breast cancer cell lines. PRR16 overexpression induced EMT in MCF7 breast cancer cells and enhances migration and invasion. To determine how PRR16 induces EMT, the binding proteins for PRR16 were screened, revealing that PRR16 binds to Abl interactor 2 (ABI2). We then investigated whether ABI2 is involved in EMT. Gene silencing of ABI2 induces EMT, leading to enhanced migration and invasion. ABI2 is a gene that codes for a protein that interacts with ABL proto-oncogene 1 (ABL1) kinase. Therefore, we investigated whether the change in ABI2 expression affected the activation of ABL1 kinase. The knockdown of ABI2 and PRR16 overexpression increased the phosphorylation of Y412 in ABL1 kinase. Our results suggest that PRR16 may be involved in EMT by binding to ABI2 and interfering with its inhibition of ABL1 kinase. This indicates that ABL1 kinase inhibitors may be potential therapeutic agents for the treatment of PRR16-related breast cancer.

Development of transgenic models susceptible and resistant to SARS-CoV-2 infection in FVB background mice.

Coronavirus disease (COVID-19), caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), is currently spreading globally. To overcome the COVID-19 pandemic, preclinical evaluations of vaccines and therapeutics using K18-hACE2 and CAG-hACE2 transgenic mice are ongoing. However, a comparative study on SARS-CoV-2 infection between K18-hACE2 and CAG-hACE2 mice has not been published. In this study, we compared the susceptibility and resistance to SARS-CoV-2 infection between two strains of transgenic mice, which were generated in FVB background mice. K18-hACE2 mice exhibited severe weight loss with definitive lethality, but CAG-hACE2 mice survived; and differences were observed in the lung, spleen, cerebrum, cerebellum, and small intestine. A higher viral titer was detected in the lungs, cerebrums, and cerebellums of K18-hACE2 mice than in the lungs of CAG-hACE2 mice. Severe pneumonia was observed in histopathological findings in K18-hACE2, and mild pneumonia was observed in CAG-hACE2. Atrophy of the splenic white pulp and reduction of spleen weight was observed, and hyperplasia of goblet cells with villi atrophy of the small intestine was observed in K18-hACE2 mice compared to CAG-hACE2 mice. These results indicate that K18-hACE2 mice are relatively susceptible to SARS-CoV-2 and that CAG-hACE2 mice are resistant to SARS-CoV-2. Based on these lineage-specific sensitivities, we suggest that K18-hACE2 mouse is suitable for highly susceptible model of SARS-CoV-2, and CAG-hACE2 mouse is suitable for mild susceptible model of SARS-CoV-2 infection.

Loss of EMP2 Inhibits Melanogenesis of MNT1 Melanoma Cells via Regulation of TRP-2.

Melanogenesis is the production of melanin from tyrosine by a series of enzyme-catalyzed reactions, in which tyrosinase and DOPA oxidase play key roles. The melanin content in the skin determines skin pigmentation. Abnormalities in skin pigmentation lead to various skin pigmentation disorders. Recent research has shown that the expression of EMP2 is much lower in melanoma than in normal melanocytes, but its role in melanogenesis has not yet been elucidated. Therefore, we investigated the role of EMP2 in the melanogenesis of MNT1 human melanoma cells. We examined TRP-1, TRP-2, and TYR expression levels during melanogenesis in MNT1 melanoma cells by gene silencing of EMP2. Western blot and RT-PCR results confirmed that the expression levels of TYR and TRP-2 were decreased when EMP2 expression was knocked down by EMP2 siRNA in MNT1 cells, and these changes were reversed when EMP2 was overexpressed. We verified the EMP2 gene was knocked out of the cell line (EMP2 CRISPR/Cas9) by using a CRISPR/Cas9 system and found that the expression levels of TRP-2 and TYR were significantly lower in the EMP2 CRISPR/Cas9 cell lines. Loss of EMP2 also reduced migration and invasion of MNT1 melanoma cells. In addition, the melanosome transfer from the melanocytes to keratinocytes in the EMP2 KO cells cocultured with keratinocytes was reduced compared to the cells in the control coculture group. In conclusion, these results suggest that EMP2 is involved in melanogenesis via the regulation of TRP-2 expression.

Corrigendum: Mouse models of lung-specific SARS-CoV-2 infection with moderate pathological traits.

[This corrects the article DOI: 10.3389/fimmu.2022.1055811.].

Mouse models of lung-specific SARS-CoV-2 infection with moderate pathological traits.

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.

Laboratory information management system for COVID-19 non-clinical efficacy trial data.

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.

Inference of a causal relation between low-density lipoprotein cholesterol and hypertension using mendelian randomization analysis.

BACKGROUND: It is known in some studies that higher the LDL-C, the greater the risk of developing cardiovascular disease. However, studies of the causal effects between LDL-C and hypertension are limited by their observational study design, and genetic epidemiology studies of associations between LDL-C and hypertension are lacking, as are studies using data for Koreans. In this study, we confirmed the causal effect of LDL-C on hypertension using Korean chip data. METHOD: The epidemiology and genotype data were collected from the Korean Genome and Epidemiology Study conducted by the Korea National Institute of Health and covered 20,701 subjects. Single-nucleotide polymorphisms associated with LDL-C were selected (p-value < 5 × 10- 8) from the Global Lipids Genetics Consortium database, and Mendelian randomization analysis (MRA) was performed with counted genetic risk scores and weighted genetic risk scores (WGRSs) for 24 single-nucleotide polymorphisms. RESULT: The assumptions for MRA were statistically confirmed, and WGRSs showed a strong association with LDL-C. Interestingly, while the relationship between LDL-C and hypertension was not statistically significant in the observational study, MRA study demonstrated that the risk of hypertension increased as LDL-C increased in both men and women. CONCLUSIONS: The results of this study confirmed that the relationship between LDL-C and hypertension is greatly influenced by genetic information.