A mouse xenograft long-term replication yields a SARS-CoV-2 Delta mutant with increased lethality.

We recently established a long-term SARS-CoV-2 infection model using lung-cancer xenograft mice and identified mutations that arose in the SARS-CoV-2 genome during long-term propagation. Here, we applied our model to the SARS-CoV-2 Delta variant, which has increased transmissibility and immune escape compared with ancestral SARS-CoV-2. We observed limited mutations in SARS-CoV-2 Delta during long-term propagation, including two predominant mutations: R682W in the spike protein and L330W in the nucleocapsid protein. We analyzed two representative isolates, Delta-10 and Delta-12, with both predominant mutations and some additional mutations. Delta-10 and Delta-12 showed lower replication capacity compared with SARS-CoV-2 Delta in cultured cells; however, Delta-12 was more lethal in K18-hACE2 mice compared with SARS-CoV-2 Delta and Delta-10. Mice infected with Delta-12 had higher viral titers, more severe histopathology in the lungs, higher chemokine expression, increased astrocyte and microglia activation, and extensive neutrophil infiltration in the brain. Brain tissue hemorrhage and mild vacuolation were also observed, suggesting that the high lethality of Delta-12 was associated with lung and brain pathology. Our long-term infection model can provide mutant viruses derived from SARS-CoV-2 Delta and knowledge about the possible contributions of emergent mutations to the properties of new variants.

Analysis of SARS-CoV-2 omicron mutations that emerged during long-term replication in a lung cancer xenograft mouse model.

SARS-CoV-2 Omicron has the largest number of mutations among all the known SARS-CoV-2 variants. The presence of these mutations might explain why Omicron is more infectious and vaccines have lower efficacy to Omicron than other variants, despite lower virulence of Omicron. We recently established a long-term in vivo replication model by infecting Calu-3 xenograft tumors in immunodeficient mice with parental SARS-CoV-2 and found that various mutations occurred majorly in the spike protein during extended replication. To investigate whether there are differences in the spectrum and frequency of mutations between parental SARS-CoV-2 and Omicron, we here applied this model to Omicron. At 30 days after infection, we found that the virus was present at high titers in the tumor tissues and had developed several rare sporadic mutations, mainly in ORF1ab with additional minor spike protein mutations. Many of the mutant isolates had higher replicative activity in Calu-3 cells compared with the original SARS-CoV-2 Omicron virus, suggesting that the novel mutations contributed to increased viral replication. Serial propagation of SARS-CoV-2 Omicron in cultured Calu-3 cells resulted in several rare sporadic mutations in various viral proteins with no mutations in the spike protein. Therefore, the genome of SARS-CoV-2 Omicron seems largely stable compared with that of the parental SARS-CoV-2 during extended replication in Calu-3 cells and xenograft model. The sporadic mutations and modified growth properties observed in Omicron might explain the emergence of Omicron sublineages. However, we cannot exclude the possibility of some differences in natural infection.

In vitro and in vivo suppression of SARS-CoV-2 replication by a modified, short, cell-penetrating peptide targeting the C-terminal domain of the viral spike protein.

Peptides are promising therapeutic agents for COVID-19 because of their specificity, easy synthesis, and ability to be fine-tuned. We previously demonstrated that a cell-permeable peptide corresponding to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) Spike C-terminal domain (CD) inhibits the interaction between viral spike and nucleocapsid proteins that results in SARS-CoV-2 replication in vitro. Here, we used docking studies to design R-t-Spike CD(D), a more potent short cell-penetrating peptide composed of all D-form amino acids and evaluated its inhibitory effect against the replication of SARS-CoV-2 S clade and other variants. R-t-Spike CD(D) was internalized into Vero cells and Calu-3 cells and suppressed the replication of SARS-CoV-2 S clade, delta variant, and omicron variant with higher potency than the original peptide. In hemizygous K18-hACE2 mice, intratracheal administration of R-t-Spike CD(D) effectively delivered the peptide to the trachea and lungs, whereas intranasal administration delivered the peptide mostly to the upper respiratory system and stomach, and a small amount to the lungs. Administration by either route reduced viral loads in mouse lungs and turbinates. Furthermore, intranasally administered R-t-Spike CD(D) mitigated pathological change in the lungs and increased the survival of mice after infection with the SARS-CoV-2 S clade or delta variant. Our data suggest that R-t-Spike CD(D) has potential as a therapeutic agent against SARS-CoV-2 infection.

A pilot study of the antiviral activity of anionic and cationic polyamidoamine dendrimers against the Middle East respiratory syndrome coronavirus.

The Middle East respiratory syndrome coronavirus (MERS-CoV) is an emerging virus that causes infection with a potentially fatal outcome. Dendrimers are highly branched molecules that can be added to antiviral preparations to improve their delivery, as well as their intrinsic antiviral activity. Studies on identifying anti-MERS-CoV agents are few. Three types of polyanionic dendrimers comprising the terminal groups sodium carboxylate (generations 1.5, 2.5, 3.5, and 4.5), hydroxyl (generations 2, 3, 4, and 5), and succinamic acid (generations 2, 3, 4, and 5) and polycationic dendrimers containing primary amine (generations 2, 3, 4, and 5) were used to assess their antiviral activity with the MERS-CoV plaque inhibition assay. The hydroxyl polyanionic set showed a 17.36% to 29.75% decrease in MERS-CoV plaque formation. The most potent inhibition of MERS-CoV plaque formation was seen by G(1.5)-16COONa (40.5% inhibition), followed by G(5)-128SA (39.77% inhibition). In contrast, the cationic dendrimers were cytotoxic to Vero cells. Polyanionic dendrimers can be added to antiviral preparations to improve the delivery of antivirals, as well as the intrinsic antiviral activity.

CD83 expression regulates antibody production in response to influenza A virus infection.

BACKGROUND: CD83 is known to regulate lymphocyte maturation, activation, homeostasis, and antibody response to immunization and infection. While CD83 has a major part in B cell function, its role in influenza A virus infection has not yet been investigated. METHODS: We investigated the role of CD83 using C57BL/6J wild type mice and CD83 knockout (KO) mice after intraperitoneal administration of the influenza A/WSN/1933 virus. We analyzed cells of the peritoneal cavity, splenocytes, and cells of the bone marrow with FACS to investigate CD83 expression and cell population change in response to the virus infection. ELISA was performed with sera and peritoneal cavity fluids to detect A/WSN/1933 virus-specific IgG and the subclasses of IgG. RESULTS: FACS analysis data showed a transient but distinct induction of CD83 expression in the peritoneal B cells of wild type mice. CD83 KO mice exhibited a delayed recovery of B cells in the bone marrow after influenza virus infection and overall, a smaller T cell population compared to wild type mice. The peritoneal cavity and serum of the wild type mice contained a high titer of IgG within 14 days after infection, whereas the CD83 KO mice had a very low titer of IgG. CONCLUSIONS: These results show the importance of CD83 in lymphocytes homeostasis and antibody production during influenza A virus infection.

Anti-Bacterial Effect of CpG-DNA Involves Enhancement of the Complement Systems.

CpG-DNA activates the host immune system to resist bacterial infections. In this study, we examined the protective effect of CpG-DNA in mice against Escherichia coli (E. coli) K1 infection. Administration of CpG-DNA increased the survival of mice after E. coli K1 infection, which reduces the numbers of bacteria in the organs. Pre-injection of mice with CpG-DNA before E. coli K1 infection increased the levels of the complement C3 but not C3a and C3b. The survival of the mice after E. coli K1 infection was significantly decreased when the mice were pre-injected with the cobra venom factor (CVF) removing the complement compared to the non-CVF-treated mice group. It suggests that the complement has protective roles against E. coli K1 infection. In addition, the survival of complement-depleted mice was increased by CpG-DNA pre-administration before E. coli K1 infection. Therefore, we suggest that CpG-DNA enhances the anti-bacterial activity of the immune system by augmenting the levels of complement systems after E. coli K1 infection and triggering other factors as well. Further studies are required to investigate the functional roles of the CpG-DNA-induced complement regulation and other factors against urgent bacterial infection.

A Novel Monoclonal Antibody Targets Mucin1 and Attenuates Growth in Pancreatic Cancer Model.

Mucin1 (MUC1) is a highly glycosylated transmembrane protein that plays a crucial role in the lubrication and protection of normal epithelial cells. However, MUC1 has emerged as a potential target for cancer therapy because it is overexpressed and functions in several types of cancers. Recently, we produced a monoclonal antibody (the anti-hMUC1 antibody) specific to the extracellular region of the MUC1 subunit MUC1-C to evaluate the utility of using anti-MUC1 antibodies in pancreatic cancer models. The anti-hMUC1 antibody recognized the MUC1-C protein in pancreatic cancer cells. Based on immunostaining and confocal image analyses, the anti-hMUC1 antibody initially bound to the cell membrane then was internalized in cancer cells that express MUC1. The anti-hMUC1 antibody suppressed epidermal growth factor (EGF)-mediated extracellular signal⁻regulated kinase (ERK) phosphorylation and cyclin D1 expression. When the anti-hMUC1 antibody was injected into a xenograft mouse model and traced using an in vivo imaging system, we observed that the anti-hMUC1 antibody was localized to MUC1-expressing pancreatic tumors. Importantly, the anti-hMUC1 monoclonal antibody suppressed pancreatic tumor growth in mice. According to immunohistochemistry analysis using a pancreatic cancer tissue array and the anti-hMUC1 antibody, MUC1 was highly expressed in human pancreatic cancer tissues compared to normal tissues. Therefore, we conclude that the anti-hMUC1 antibody specifically targets MUC1 and suppresses its function in pancreatic cancer in vitro and in vivo and can be further developed as a promising targeted therapy to treat pancreatic cancer.

Impact of p16 expression in oropharyngeal cancer in the postoperative setting: the necessity of re-evaluating traditional risk stratification.

OBJECTIVE: To evaluate the impact of p16 expression as a surrogate marker of human papillomavirus status in oropharyngeal squamous cell carcinoma patients underwent surgery followed by postoperative radiotherapy. METHODS: We identified 126 consecutive patients with histologically confirmed, newly diagnosed oropharyngeal squamous cell carcinoma who received surgery followed by radiotherapy and had p16 expression data available. All patients were treated between 2001 and 2011. Patients with high-risk factors (positive surgical margin and/or extracapsular extension) or other risk factors (multiple positive lymph nodes, perineural/lymphovascular invasion) were offered postoperative radiotherapy with or without concurrent chemotherapy. RESULTS: One hundred and four (82.5%) patients were p16-positive (p16 (+)) and 22 (17.5%) were p16-negative (p16 (-)). With a median follow-up of 56 months, patients with p16 (+) oropharyngeal squamous cell carcinoma exhibited a significantly better 5-year disease-free survival (80.7% vs. 57.6%, P < 0.001) and overall survival (84.9% vs. 59.1%, P < 0.001) than those with p16 (-) tumors. The p16 (+) oropharyngeal squamous cell carcinoma with high-risk factors (n = 64) showed no difference in disease-free survival (79.7% vs. 68.3%; P = 0.531) and overall survival (82.1% vs. 76.2%; P = 0.964) between postoperative radiotherapy and postoperative radiotherapy with concurrent chemotherapy. CONCLUSIONS: Expression of p16 is a strong independent prognostic factor of survival in the postoperative setting of oropharyngeal squamous cell carcinoma. The favorable prognosis of p16 (+) oropharyngeal squamous cell carcinoma suggests a need to re-examine traditional risk stratification for determining optimal adjuvant treatment.

Plasminogen activator inhibitor-1 regulates infiltration of macrophages into melanoma via phosphorylation of FAK-Tyr9²5.

Tumor-infiltrating macrophages are potential candidates for cancer immunotherapy. However, the detailed molecular mechanism underlying macrophage infiltration into tumors is poorly understood. Based on our previous finding that plasminogen activator inhibitor-1 (PAI-1) enhances vitronectin-dependent migration of macrophages, we investigated the potential role of PAI-1 in macrophage invasion into melanoma. Experimental evidence obtained from spheroid confrontation assay clearly showed that PAI-1 overexpression significantly enhanced the invasion of RAW 264.7 cells into B16F10 melanoma. We further demonstrated that PAI-1 induces phosphorylation of focal adhesion kinase (FAK) at Tyr(925), which, in turn, mediated the invasion of macrophages into the melanoma. This work further illustrates that low-density lipoprotein receptor related-protein 1 (LRP1) is essential for PAI-1-mediated FAK phosphorylation and macrophage invasion into melanoma. In conclusion, our study demonstrates a novel role of PAI-1 in macrophage invasion into melanoma and provides insights into the underlying molecular mechanism.

The PDZ-binding motif of the avian NS1 protein affects transmission of the 2009 influenza A(H1N1) virus.

By nature of their segmented RNA genome, influenza A viruses (IAVs) have the potential to generate variants through a reassortment process. The influenza nonstructural (NS) gene is critical for a virus to counteract the antiviral responses of the host. Therefore, a newly acquired NS segment potentially determines the replication efficiency of the reassortant virus in a range of different hosts. In addition, the C-terminal PDZ-binding motif (PBM) has been suggested as a pathogenic determinant of IAVs. To gauge the pandemic potential from human and avian IAV reassortment, we assessed the replication properties of NS-reassorted viruses in cultured cells and in the lungs of mice and determined their transmissibility in guinea pigs. Compared with the recombinant A/Korea/01/2009 virus (rK09; 2009 pandemic H1N1 strain), the rK09/VN:NS virus, in which the NS gene was adopted from the A/Vietnam/1203/2004 virus (a human isolate of the highly pathogenic avian influenza H5N1 virus strains), exhibited attenuated virulence and reduced transmissibility. However, the rK09/VN:NS-PBM virus, harboring the PBM in the C-terminus of the NS1 protein, recovered the attenuated virulence of the rK09/VN:NS virus. In a guinea pig model, the rK09/VN:NS-PBM virus showed even greater transmission efficiency than the rK/09 virus. These results suggest that the PBM in the NS1 protein may determine viral persistence in the human and avian IAV interface.

Inhibition of Pseudomonas aeruginosa with a recombinant RNA-based viral vector expressing human ß-defensin 4.

BACKGROUND: Harassed with extensive epithelial burn wounds, patients can be affected by complications, such as infection, hypovolemic shock, hypothermia, and respiratory failure. Immediate first aid and followed supportive cares are critical for the prevention of severe complications. However, secondary bacterial infection is hard to be controlled in burn patients, and Pseudomonas aeruginosa (P. aeruginosa) is one of the top listed pathogens perturbing burn wounds beyond the antibiotics spectrum. RESULTS: To find the way for efficacious protection from the pseudomonas-mediated complications in burn patients, we assessed the in vitro and in vivo inhibitory values of human ß-defensin 4 (hBD4), which is known as a member of the cationic, antimicrobial peptides found in human cells of many kinds. The Newcastle disease virus (NDV) was used as a viral vector for the expression of hBD4 in burn wounds. Expressed from the recombinant NDV (rNDV-hBD4), hBD4 effectively inhibited the pseudomonal growths in cell culture media. In a mouse model, severely burn-injured skin was recovered by the direct installation of the rNDV-hBD4 infected cells in the burn wounds whereas that of control mice remained severely damaged. CONCLUSIONS: We suggest that the application of hBD4 may protect burn patients from secondary pseudomonal infection and provide a therapeutic potential for burn wound treatment.

Contribution of HSP90 Cleavage to the Cytotoxic Effect of Suberoylanilide Hydroxamic Acid In Vivo and the Involvement of TXNIP in HSP90 Cleavage.

Heat shock protein (HSP) 90 is expressed in most living organisms, and several client proteins of HSP90 are necessary for cancer cell survival and growth. Previously, we found that HSP90 was cleaved by histone deacetylase (HDAC) inhibitors and proteasome inhibitors, and the cleavage of HSP90 contributes to their cytotoxicity in K562 leukemia cells. In this study, we first established mouse xenograft models with K562 cells expressing the wild-type or cleavage-resistant mutant HSP90ß and found that the suppression of tumor growth by the HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) was interrupted by the mutation inhibiting the HSP90 cleavage in vivo. Next, we investigated the possible function of thioredoxin interacting protein (TXNIP) in the HSP90 cleavage induced by SAHA. TXNIP is a negative regulator for thioredoxin, an antioxidant protein. SAHA transcriptionally induced the expression of TXNIP in K562 cells. HSP90 cleavage was induced by SAHA also in the thymocytes of normal mice and suppressed by an anti-oxidant and pan-caspase inhibitor. When the thymocytes from the TXNIP knockout mice and their wild-type littermate control mice were treated with SAHA, the HSP90 cleavage was detected in the thymocytes of the littermate controls but suppressed in those of the TXNIP knockout mice suggesting the requirement of TXNIP for HSP90 cleavage. We additionally found that HSP90 cleavage was induced by actinomycin D, ß-mercaptoethanol, and p38 MAPK inhibitor PD169316 suggesting its prevalence. Taken together, we suggest that HSP90 cleavage occurs also in vivo and contributes to the anti-cancer activity of various drugs in a TXNIP-dependent manner.

Analysis of SARS-CoV-2 Mutations after Nirmatrelvir Treatment in a Lung Cancer Xenograft Mouse Model.

Paxlovid is the first approved oral treatment for coronavirus disease 2019 and includes nirmatrelvir, a protease inhibitor targeting the main protease (Mpro) of SARS-CoV-2, as one of the key components. While some specific mutations emerged in Mpro were revealed to significantly reduce viral susceptibility to nirmatrelvir in vitro, there is no report regarding resistance to nirmatrelvir in patients and animal models for SARS-CoV-2 infection yet. We recently developed xenograft tumors derived from Calu-3 cells in immunodeficient mice and demonstrated extended replication of SARS-CoV-2 in the tumors. In this study, we investigated the effect of nirmatrelvir administration on SARS-CoV-2 replication. Treatment with nirmatrelvir after virus infection significantly reduced the replication of the parental SARS-CoV-2 and SARS-CoV-2 Omicron at 5 days post-infection (dpi). However, the virus titers were completely recovered at the time points of 15 and 30 dpi. The virus genomes in the tumors at 30 dpi were analyzed to investigate whether nirmatrelvir-resistant mutant viruses had emerged during the extended replication of SARS-CoV-2. Various mutations in several genes including ORF1ab, ORF3a, ORF7a, ORF7b, ORF8, and N occurred in the SARS-CoV-2 genome; however, no mutations were induced in the Mpro sequence by a single round of nirmatrelvir treatment, and none were observed even after two rounds of treatment. The parental SARS-CoV-2 and its sublineage isolates showed similar IC50 values of nirmatrelvir in Vero E6 cells. Therefore, it is probable that inducing viral resistance to nirmatrelvir in vivo is challenging differently from in vitro passage.

Matrix metalloproteinase 9 (MMP-9)-dependent processing of ßig-h3 protein regulates cell migration, invasion, and adhesion.

Cell migration is critically involved in inflammation, cancer, and development. In this study, transforming growth factor-ß-induced protein (ßig-h3) was identified as a substrate of matrix metalloproteinase-9 (MMP-9) by site-directed mutagenesis. ßig-h3 has two cleavage sites with the consensus sequence Pro-Xaa-Xaa-Hy-(Ser/Thr) (Hy is a hydrophobic amino acid) (PGSFT beginning at amino acid 135 and PPMGT beginning at amino acid 501). Using recombinant human ßig-h3 and MMP-9, ßig-h3 from ßig-h3-transfected HEK293F cells, and MMP-9 from MMP-9-transfected HEK293F cells, human macrophages, and neutrophils, we found that MMP-9 proteolytically cleaves ßig-h3. Cleavage leads to the loss of its adhesive property and its release from extracellular matrix proteins, collagen IV, and fibronectin. Spheroids formed by increased cell-cell interactions were observed in ßig-h3-transfected HEK293F cells but not in vehicle-transfected HEK293F cells. In human glioma U87MG cells, MMP-9 constitutive overexpression resulted in endogenous ßig-h3 cleavage. ßig-h3 cleavage by MMP-9 led to increased cell invasion, and ßig-h3 knockdown also resulted in increased cell invasion. The ßig-h3 fragment cleaved by MMP-9 could bind to the surface of macrophages, and it may play a role as a peptide chemoattractant by inducing macrophage migration via focal adhesion kinase/Src-mediated signal activation. Thus, intact ßig-h3 is responsible for cell migration inhibition, cell-cell contact, and cell-extracellular matrix interaction. Experimental evidence indicates that MMP-9-cleaved ßig-h3 plays a role in MMP-9-mediated tumor cell and macrophage migration.

Prophylactic effect of a peptide vaccine targeting TM4SF5 against colon cancer in a mouse model.

Expression of transmembrane 4 superfamily member 5 protein (TM4SF5) was implicated in hepatocellular carcinoma (HCC) and colon cancer. Previously, we have shown that immunization with TM4SF5 peptide-CpG-DNA-liposome complex induces production of TM4SF5-specific antibodies and protects mice from HCC progression in an allograft model. Here, we confirmed expression of TM4SF5 in the mouse colon cancer cell line CT-26 and found that anti-TM4SF5 antibody inhibits growth of CT-26 cells. We then immunized mice with TM4SF5 peptide-CpG-DNA-liposome complex and transplanted CT-26 cells to investigate the vaccination effects. Robust production of TM4SF5-specific antibodies was induced by challenge with CT-26 cells and the tumor growth was significantly suppressed in the immunized mice. The peptide vaccine targeting TM4SF5 consequently showed a prophylactic effect against colon cancer development in a mouse model. These results suggest that the peptide vaccine can be potentially applied in humans to treat colon cancer.

Protective Effects of Oleic Acid Against Palmitic Acid-Induced Apoptosis in Pancreatic AR42J Cells and Its Mechanisms.

Palmitic acid (PAM), one of the most common saturated fatty acid (SFA) in animals and plants, has been shown to induce apoptosis in exocrine pancreatic AR42J cells. In this study, we investigated cellular mechanisms underlying protective effects of oleic acid (OLA) against the lipotoxic actions of PAM in AR42J cells. Exposure of cells to long-chain SFA induced apoptotic cell death determined by MTT cell viability assay and Hoechst staining. Co-treatment of OLA with PAM markedly protected cells against PAM-induced apoptosis. OLA significantly attenuated the PAM-induced increase in the levels of pro-apoptotic Bak protein, cleaved forms of apoptotic proteins (caspase-3, PARP). On the contrary, OLA restored the decreased levels of anti-apoptotic Bcl-2 family proteins (Bcl-2, Bcl-xL, and Mcl-1) in PAM-treated cells. OLA also induced up-regulation of the mRNA expression of Dgat2 and Cpt1 genes which are involved in triacylglycerol (TAG) synthesis and mitochondrial ß-oxidation, respectively. Intracellular TAG accumulation was increased by OLA supplementation in accordance with enhanced expression of Dgat2 gene. These results indicate that restoration of anti-apoptotic/pro-apoptotic protein balance from apoptosis toward cell survival is involved in the cytoprotective effects of OLA against PAM-induced apoptosis in pancreatic AR42J cells. In addition, OLA-induced increase in TAG accumulation and up-regulation of Dgat2 and Cpt1 gene expressions may be possibly associated in part with the ability of OLA to protect cells from deleterious actions of PAM.

SARS-CoV-2 infection induces expression and secretion of lipocalin-2 and regulates iron in a human lung cancer xenograft model.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection leads to various clinical symptoms including anemia. Lipocalin-2 has various biological functions, including defense against bacterial infections through iron sequestration, and it serves as a biomarker for kidney injury. In a human protein array, we observed increased lipocalin-2 expression due to parental SARS-CoV-2 infection in the Calu-3 human lung cancer cell line. The secretion of lipocalin-2 was also elevated in response to parental SARS-CoV-2 infection, and the SARS-CoV-2 Alpha, Beta, and Delta variants similarly induced this phenomenon. In a Calu-3 implanted mouse xenograft model, parental SARSCoV- 2 and Delta variant induced lipocalin-2 expression and secretion. Additionally, the iron concentration increased in the Calu-3 tumor tissues and decreased in the serum due to infection. In conclusion, SARS-CoV-2 infection induces the production and secretion of lipocalin-2, potentially resulting in a decrease in iron concentration in serum. Because the concentration of iron ions in the blood is associated with anemia, this phenomenon could contribute to developing anemia in COVID-19 patients. [BMB Reports 2023; 56(12): 669-674].

Analysis of spike protein variants evolved in a novel in vivo long-term replication model for SARS-CoV-2.

Introduction: The spectrum of SARS-CoV-2 mutations have increased over time, resulting in the emergence of several variants of concern. Persistent infection is assumed to be involved in the evolution of the variants. Calu-3 human lung cancer cells persistently grow without apoptosis and release low virus titers after infection. Methods: We established a novel in vivo long-term replication model using xenografts of Calu-3 human lung cancer cells in immunodeficient mice. Virus replication in the tumor was monitored for 30 days and occurrence of mutations in the viral genome was determined by whole-genome deep sequencing. Viral isolates with mutations were selected after plaque forming assays and their properties were determined in cells and in K18-hACE2 mice. Results: After infection with parental SARS-CoV-2, viruses were found in the tumor tissues for up to 30 days and acquired various mutations, predominantly in the spike (S) protein, some of which increased while others fluctuated for 30 days. Three viral isolates with different combination of mutations produced higher virus titers than the parental virus in Calu-3 cells without cytopathic effects. In K18-hACE2 mice, the variants were less lethal than the parental virus. Infection with each variant induced production of cross-reactive antibodies to the receptor binding domain of parental SARS-CoV-2 S protein and provided protective immunity against subsequent challenge with parental virus. Discussion: These results suggest that most of the SARS-CoV-2 variants acquired mutations promoting host adaptation in the Calu-3 xenograft mice. This model can be used in the future to further study SARS-CoV-2 variants upon long-term replication in vivo.

Prevention of hypoglycemia-induced neuronal death by minocycline.

Diabetic patients who attempt strict management of blood glucose levels frequently experience hypoglycemia. Severe and prolonged hypoglycemia causes neuronal death and cognitive impairment. There is no effective tool for prevention of these unwanted clinical sequelae. Minocycline, a second-generation tetracycline derivative, has been recognized as an anti-inflammatory and neuroprotective agent in several animal models such as stroke and traumatic brain injury. In the present study, we tested whether minocycline also has protective effects on hypoglycemia-induced neuronal death and cognitive impairment. To test our hypothesis we used an animal model of insulin-induced acute hypoglycemia. Minocycline was injected intraperitoneally at 6 hours after hypoglycemia/glucose reperfusion and injected once per day for the following 1 week. Histological evaluation for neuronal death and microglial activation was performed from 1 day to 1 week after hypoglycemia. Cognitive evaluation was conducted 6 weeks after hypoglycemia. Microglial activation began to be evident in the hippocampal area at 1 day after hypoglycemia and persisted for 1 week. Minocycline injection significantly reduced hypoglycemia-induced microglial activation and myeloperoxidase (MPO) immunoreactivity. Neuronal death was significantly reduced by minocycline treatment when evaluated at 1 week after hypoglycemia. Hypoglycemia-induced cognitive impairment is also significantly prevented by the same minocycline regimen when subjects were evaluated at 6 weeks after hypoglycemia. Therefore, these results suggest that delayed treatment (6 hours post-insult) with minocycline protects against microglial activation, neuronal death and cognitive impairment caused by severe hypoglycemia. The present study suggests that minocycline has therapeutic potential to prevent hypoglycemia-induced brain injury in diabetic patients.

CXC chemokine ligand 2 induced by receptor activator of NF-kappa B ligand enhances osteoclastogenesis.

CXCL2 has been known to regulate immune functions mainly by chemo-attracting neutrophils. In this study, we show that CXCL2 can be induced by receptor activator of NF-kappaB ligand, the osteoclast (OC) differentiation factor, through JNK and NF-kappaB signaling pathways in OC precursor cells. CXCL2 in turn enhanced the proliferation of OC precursor cells of bone marrow-derived macrophages (BMMs) through the activation of ERK. Knockdown of CXCL2 inhibited both the proliferation of and the ERK activation in BMMs. During osteoclastogenesis CXCL2 stimulated the adhesion and the migration of BMMs. Moreover, the formation of OCs from BMMs was significantly increased on treatment with CXCL2. Conversely, the CXCL2 antagonist repertaxin and a CXCL2 neutralizing Ab potently reduced receptor activator of NF-kappaB ligand-induced osteoclastogenesis. Furthermore, CXCL2 evoked fulminant bone erosion in the in vivo mouse experiments. Finally, prominent upregulation of CXCL2 was detected in synovial fluids and sera from rheumatoid arthritis patients, suggesting a potential involvement of CXCL2-mediated osteoclastogenesis in rheumatoid arthritis-associated bone destruction. Thus, CXCL2 is a novel therapeutic target for inflammatory bone destructive diseases.