TLR9 and STING agonists cooperatively boost the immune response to SARS-CoV-2 RBD vaccine through an increased germinal center B cell response and reshaped T helper responses.

Vaccines are a powerful medical intervention for preventing epidemic diseases. Efficient inactivated or protein vaccines typically rely on an effective adjuvant to elicit an immune response and boost vaccine activity. In this study, we investigated the adjuvant activities of combinations of Toll-like receptor 9 (TLR9) and stimulator of interferon genes (STING) agonists in a SARS-CoV-2 receptor binding domain protein vaccine. Adjuvants formulated with a TLR9 agonist, CpG-2722, with various cyclic dinucleotides (CDNs) that are STING agonists increased germinal center B cell response and elicited humoral immune responses in immunized mice. An adjuvant containing CpG-2722 and 2'3'-c-di-AM(PS)2 effectively boosted the immune response to both intramuscularly and intranasally administrated vaccines. Vaccines adjuvanted with CpG-2722 or 2'3'-c-di-AM(PS)2 alone were capable of inducing an immune response, but a cooperative adjuvant effect was observed when both were combined. CpG-2722 induced antigen-dependent T helper (Th)1 and Th17 responses, while 2'3'-c-di-AM(PS)2 induced a Th2 response. The combination of CpG-2722 and 2'3'-c-di-AM(PS)2 generated a distinct antigen-dependent Th response profile characterized by higher Th1 and Th17, but lower Th2 responses. In dendritic cells, CpG-2722 and 2'3'-c-di-AM(PS)2 showed a cooperative effect on inducing expression of molecules critical for T cell activation. CpG-2722 and 2'3'-c-di-AM(PS)2 have distinct cytokine inducing profiles in different cell populations. The combination of these two agonists enhanced the expression of cytokines for Th1 and Th17 responses and suppressed the expression of cytokines for Th2 response in these cells. Thus, the antigen-dependent Th responses observed in the animals immunized with different vaccines were shaped by the antigen-independent cytokine-inducing profiles of their adjuvant. The expanded targeting cell populations, the increased germinal center B cell response, and reshaped T helper responses are the molecular bases for the cooperative adjuvant effect of the combination of TLR9 and STING agonists.

Furin and TMPRSS2 Resistant Spike Induces Robust Humoral and Cellular Immunity Against SARS-CoV-2 Lethal Infection.

An effective COVID-19 vaccine against broad SARS-CoV-2 variants is still an unmet need. In the study, the vesicular stomatitis virus (VSV)-based vector was used to express the SARS-CoV-2 Spike protein to identify better vaccine designs. The replication-competent of the recombinant VSV-spike virus with C-terminal 19 amino acid truncation (SΔ19 Rep) was generated. A single dose of SΔ19 Rep intranasal vaccination is sufficient to induce protective immunity against SARS-CoV-2 infection in hamsters. All the clones isolated from the SΔ19 Rep virus contained R682G mutation located at the Furin cleavage site. An additional S813Y mutation close to the TMPRSS2 cleavage site was identified in some clones. The enzymatic processing of S protein was blocked by these mutations. The vaccination of the R682G-S813Y virus produced a high antibody response against S protein and a robust S protein-specific CD8+ T cell response. The vaccinated animals were protected from the lethal SARS-CoV-2 (delta variant) challenge. The S antigen with resistance to enzymatic processes by Furin and TMPRSS2 will provide better immunogenicity for vaccine design.

Lipocalin-2 mediates the rejection of neural transplants.

Lipocalin-2 (LCN2) has been implicated in promoting apoptosis and neuroinflammation in neurological disorders; however, its role in neural transplantation remains unknown. In this study, we cultured and differentiated Lund human mesencephalic (LUHMES) cells into human dopaminergic-like neurons and found that LCN2 mRNA was progressively induced in mouse brain after the intrastriatal transplantation of human dopaminergic-like neurons. The induction of LCN2 protein was detected in a subset of astrocytes and neutrophils infiltrating the core of the engrafted sites, but not in neurons and microglia. LCN2-immunoreactive astrocytes within the engrafted sites expressed lower levels of A1 and A2 astrocytic markers. Recruitment of microglia, neutrophils, and monocytes after transplantation was attenuated in LCN2 deficiency mice. The expression of M2 microglial markers was significantly elevated and survival of engrafted neurons was markedly improved after transplantation in LCN2 deficiency mice. Brain type organic cation transporter (BOCT), the cell surface receptor for LCN2, was induced in dopaminergic-like neurons after differentiation, and treatment with recombinant LCN2 protein directly induced apoptosis in dopaminergic-like neurons in a dose-dependent manner. Our results, therefore, suggested that LCN2 is a neurotoxic factor for the engrafted neurons and a modulator of neuroinflammation. LCN2 inhibition may be useful in reducing rejection after neural transplantation.

Differential dynamics of hepatic protein expressions with long-term cultivated hepatitis C virus infection.

BACKGROUND: The liver maintains blood chemical homeostasis by active uptake and secretion through endocytosis, exocytosis, and intracellular trafficking between the plasma and intracellular membranes. Hepatitis C virus (HCV) infection affects the host membrane architecture and might thus impair the regulation of the cellular transportation machinery. Additionally, the hepatic expressions of differential protein dynamics with long-term HCV infection remain fully recover. METHODS: In this study, comparative proteomic analysis was performed in HCV-infected and mock-control Huh7 cells according to the viral dynamics of exponential, plateau, declined, and silencing phases at the acute stage, and the chronic stage. The proteins with <0.8-fold and ≥1.25-fold changes in expression were analyzed using functional pathway clustering prediction. RESULTS: The combined experimental repetitions identified full-spectrum cellular proteins in each of 5 sample sets from acute exponential, plateau, declined, and silencing phases, and the chronic stage. The clustering results revealed that HCV infection might differentiate regulatory pathways involving extracellular exosome, cadherin, melanosome, and RNA binding. Overall host proteins in HCV-infected cells exhibited kinetic pattern 1, in which cellular expression was downregulated from the acute exponential to plateau phases, reached a nadir, and was then elevated at the chronic stage. The proteins involved in the membrane-budding pathway exhibited kinetic pattern 2, in which their expressions were distinctly downregulated at the chronic stage. CONCLUSION: The current comparative proteomics revealed the differential regulatory effects of HCV infection on host intracellular transport functional pathways, which might contribute to the pathogenic mechanisms of HCV in hepatocytes that sustain long-term infection.

Viral dynamics of persistent hepatitis C virus infection in high-sensitive reporter cells resemble patient's viremia.

BACKGROUND: Hepatitis C virus (HCV) infection has a high persistence rate in patients. Although immune cells play a central role in determining the outcomes of HCV infection, the liver is crucial in controlling HCV activity from acute to chronic stages. This investigation grew HCV from a long-term cell culture, and provided an experimental model for studies on HCV persistence in hepatocytes. METHODS: Huh7.5 cells implanted with the NS3/4 protease-based secreted alkaline phosphatase (SEAP) reporter were infected with JFH-1 HCV (moiety of infection = 0.01) and incubated for over 130 days. RESULTS: The viral activity was obtained by sampling supernatant continuously for SEAP activity measurement. Combined with extracellular and intracellular HCV-RNAs and viral infectivity assays, the experimental results exhibited in vitro viral dynamics resembling the patients' viremia pattern from acute to chronic infections. The HCV in acute infection comprised exponential accumulation (week 1), plateau (week 2), declining production (weeks 3-4) and silencing (weeks 5-14) phases, and were then reactivated at the onset of chronic infection (after week 15). The HCV-infected cells grew more slowly than the mock controls, and exhibited a prominent decrease of cell growth rate and increase of early apoptosis in the declining-to-silencing phase transition, suggesting that fitness selection might occur as the infected cells moved across the boundary of active to occult viral activity. CONCLUSION: Cultivated HCV in the highly sensitive NS3/4-based SEAP reporter cells could establish persistence, which might mimic the viral dynamics from acute to chronic infections in hepatitis C patients.

Identifying Chinese Herbal Medicine Network for Endometriosis: Implications from a Population-Based Database in Taiwan.

BACKGROUND: Endometriosis is a common but bothersome gynecological disease, and Chinese herbal medicine (CHM) is used for treating endometriosis. The aim of this study is to explore CHM network and core treatments for endometriosis by analyzing nationwide CHM prescription database. METHODS: From 1998 to 2013, the CHM prescriptions made primarily for endometriosis among women diagnosed with endometriosis (ICD-9-CM code: 671) by gynecologists during their reproductive age were collected. CHM network analysis was then carried out by using association rule mining and social network analysis. RESULTS: A total of 12,986 CHM prescriptions made for endometriosis were analyzed. There were 556 kinds of CHM ever used, and, in average, each prescription was composed of 6.2 CHMs. Gui-Zhi-Fu-Ling-Wan (GZFLW) was used most frequently, followed by Cyperus rotundus (28.1% and 18.8% of all prescriptions, resp.). Additionally, the combination of Cyperus rotundus with GZFLW (8.0%) was the most frequently used combination of two CHMs. CHM network showed that GZFLW was the core CHM for endometriosis and graphically demonstrated the extensive coverage of TCM syndromes and pathogenesis of endometriosis. CONCLUSIONS: CHM network provides graphical demonstration and summary of commonly used CHMs for endometriosis, and further studies are warranted based on these findings.

Lipoprotein lipase liberates free fatty acids to inhibit HCV infection and prevent hepatic lipid accumulation.

Lipoprotein lipase (LPL) has been identified as an anti-hepatitis C virus (HCV) host factor, but the cellular mechanism remains elusive. Here, we investigated the cellular mechanism of LPL involving in anti-HCV. The functional activation of peroxisome proliferator-activated receptor (PPAR) α signal by LPL transducing into hepatocytes was investigated in HCV-infected cells, primary human hepatocytes, and in HCV-core transgenic mice. The result showed that the levels of transcriptional transactivity and nuclear translocation of PPARα in Huh7 cells and primary human hepatocytes were elevated by physiologically ranged LPL treatment of either very-low density lipoprotein or HCV particles. The LPL-induced hepatic PPARα activation was weakened by blocking the LPL enzymatic activity, and by preventing the cellular uptake of free unsaturated fatty acids with either albumin chelator or silencing of CD36 translocase. The knockdowns of PPARα and CD36 reversed the LPL-mediated suppression of HCV infection. Furthermore, treatment with LPL, like the direct activation of PPARα, not only reduced the levels of apolipoproteins B, E, and J, which are involved in assembly and release of HCV virions, but also alleviated hepatic lipid accumulation induced by core protein. HCV-core transgenic mice exhibited more hepatic miR-27b, which negatively regulates PPARα expression, than did the wild-type controls. The induction of LPL activity by fasting in the core transgenic mice activated PPARα downstream target genes that are involved in fatty acid ß-oxidation. Taken together, our study reveals dual beneficial outcomes of LPL in anti-HCV and anti-steatosis and shed light on the control of chronic hepatitis C in relation to LPL modulators.

Ca2+ signaling in cytoskeletal reorganization, cell migration, and cancer metastasis.

Proper control of Ca(2+) signaling is mandatory for effective cell migration, which is critical for embryonic development, wound healing, and cancer metastasis. However, how Ca(2+) coordinates structural components and signaling molecules for proper cell motility had remained elusive. With the advance of fluorescent live-cell Ca(2+) imaging in recent years, we gradually understand how Ca(2+) is regulated spatially and temporally in migrating cells, driving polarization, protrusion, retraction, and adhesion at the right place and right time. Here we give an overview about how cells create local Ca(2+) pulses near the leading edge, maintain cytosolic Ca(2+) gradient from back to front, and restore Ca(2+) depletion for persistent cell motility. Differential roles of Ca(2+) in regulating various effectors and the interaction of roles of Ca(2+) signaling with other pathways during migration are also discussed. Such information might suggest a new direction to control cancer metastasis by manipulating Ca(2+) and its associating signaling molecules in a judicious manner.

Fluoxetine a novel anti-hepatitis C virus agent via ROS-, JNK-, and PPARß/γ-dependent pathways.

More than 20% of chronic hepatitis C (CHC) patients receiving interferon-alpha (IFN-α)-based anti-hepatitis C virus (HCV) therapy experienced significant depression, which was relieved by treatment with fluoxetine. However, whether and how fluoxetine affected directly the anti-HCV therapy remained unclear. Here, we demonstrated that fluoxetine inhibited HCV infection and blocked the production of reactive oxygen species (ROS) and lipid accumulation in Huh7.5 cells. Fluoxetine facilitated the IFN-α-mediated antiviral actions via activations of signal transducer and activator of transcription (STAT)-1 and c-Jun amino-terminal kinases (JNK). Alternatively, fluoxetine elevated peroxisome proliferator-activated receptor (PPAR) response element activity under HCV infection. The inhibitory effects of fluoxetine on HCV infection and lipid accumulation, but not production of ROS, were partially reversed by the PPAR-ß, -γ, and JNK antagonists. Furthermore, fluoxetine intervention to the IFN-α-2b regimen facilitated to reduce HCV titer and alanine transaminase level for CHC patients. Therefore, fluoxetine intervention to the IFN-α-2b regimen improved the efficacy of anti-HCV treatment, which might be related to blockades of ROS generation and lipid accumulation and activation of host antiviral JNK/STAT-1 and PPARß/γ signals.

Apolipoprotein J, a glucose-upregulated molecular chaperone, stabilizes core and NS5A to promote infectious hepatitis C virus virion production.

BACKGROUND & AIMS: Hepatitis C virus (HCV) infection leads to glucose abnormality. HCV depends on lipid droplets (LDs) and very-low density lipoproteins for assembly/releasing; however, the components and locations for this process remain unidentified. Apolipoprotein J (ApoJ), upregulated by glucose, functions as Golgi chaperone of secreted proteins and resides abundantly in very-low density lipoproteins. This study investigates the interplay between glucose, ApoJ and HCV virion production. METHODS: The effects of high glucose on ApoJ expression and HCV production were evaluated with cultivated HuH7.5, primary human hepatocytes, and in treatment naive chronic hepatitis C patients. How ApoJ affects HCV lifecycle was assessed using siRNA knockdown strategy in JFH1 infected and subgenomic replicon cells. The interactions and locations of ApoJ with viral and host components were examined by immunoprecipitation, immunofluorescence and subcellular fractionation experiments. RESULTS: HCV infection increased ApoJ expression, which in parallel with HCV infectivity was additionally elevated with high glucose treatment. Serum ApoJ correlated positively with fasting blood glucose concentration and HCV-RNA titre in patients. ApoJ silencing reduced intracellular and extracellular HCV infectivity and extracellular HCV-RNA, but accumulated intracellular HCV-RNA in HCV-infected cells. ApoJ interacted with HCV core and NS5A and stabilized the dual protein complex. HCV infection dispersed cytoplasmic ApoJ from the compact zones of the Golgi to encircle LDs, where co-localization of the core, NS5A, HCV-RNA, subcellular markers for LDs, endoplasmic reticulum (ER), Golgi, and membrane contact sites occurred. CONCLUSIONS: ApoJ facilitates infectious HCV particle production via stabilization of core/NS5A, which might surround LDs at the ER-Golgi membrane contact site.