Uricase-based drugs excel at treating refractory hyperuricemia and tumor lysis syndrome by directly degrading uric acid but are limited by immunogenicity. Here, we engineered RAW264.7 macrophages with ectopic co-expression of Aspergillus flavus uricase and murine urate anion transporter 1 (URAT1), forming a "transport-degradation" system: URAT1 actively transports uric acid into cells for intracellular degradation. Recombinant lentiviral vectors carrying target genes were transfected into RAW264.7 cells, followed by puromycin screening. In vitro assays showed that the engineered macrophages nearly completely degraded uric acid (from 556.0 ± 37.0 μmol/L to 0.7 ± 0.6 μmol/L) at 72 h. URAT1 inhibition with benzbromarone abolished uric acid degradation in URAT1-expressing cells. In both acute dietary-induced and chronic genetic hyperuricemic mouse models, RAW-afUri-URAT1 exerted robust and sustained uric acid-lowering activity, maintaining serum uric acid at 77.14 ± 37.48 μmol/L on day 16 in yeast extract gavaged mice and normalizing serum uric acid to 76.2 ± 15.9 μmol/L in liver uricase conditional knockout mice, both significantly superior to the rebound levels observed in mice treated with Rasburicase (143.19 ± 38.21 μmol/L and 142.4 ± 17.4 μmol/L, respectively; P < 0.05). Safety assessments in dietary-induced hyperuricemia mice showed no obvious abnormalities in liver or renal function, and significantly reduced hyperuricemia-related production of inflammatory cytokines (IL-1β, IL-6, TNF-α), Immunogenicity assays showed undetectable anti-uricase antibodies in RAW-afUri-URAT1 treated mice but high level of antibodies in rasburicase treated mice. This engineered macrophage system shows potent, durable uric acid-lowering efficacy, with low immunogenicity and good biosafety, offering a promising strategy for hyperuricemia therapy.

A core complex with transcription factors (TFs) TAL1/TCF3/GATA2 and adaptors LMO2/LDB1 lies at the top of the hematopoietic transcriptional hierarchy. The mechanism(s) underlying the expression of these components remain elusive. Adaptor RACK1 interacts with multiple TFs and modulates their activation and/or stability. However, a role of RACK1 in the transcriptional control of hematopoietic stem cell (HSC) fates hasn’t been disclosed. Here, we report that RACK1 is expressed across various hematopoietic cell types. Adulthood Rack1 deletion in type I interferon- (IFN-I)-responsive cells leads to rapid and profound hematopoietic failure and HSC loss. HSC exhaustion upon adulthood Rack1 deletion results from cell-intrinsic defects with massive apoptosis. Single-cell RNA sequencing indicates that adulthood Rack1 deletion in IFN-I-responsive cells leads to aberrant lineage-geneset-scores of transcriptional HSCs and the emergence of stressed HSCs. Furthermore, prenatal deletion of Rack1 in hematopoietic cells results in reduced and defective HSCs in the fetal liver. Mechanistically, RACK1 prevents HSC loss through maintaining the protein level of LDB1. The direct interaction between RACK1 and LDB1 suppressing its ubiquitination and subsequent degradation, thereby stabilizes LDB1. Therefore, RACK1 maintains adult and fetal mouse HSCs through, at least partially, directly binding to and stabilizing LDB1.

Pathogenic flaviviruses are predominantly the pathogens of emerging and re-emerging infectious diseases, which have caused multiple public health emergencies globally and pose a serious threat to human health and social development. Although significant achievements have been made in vaccine research, issues such as limited protective effects and virulence reversion persist, making the development of novel vaccines against pathogenic flaviviruses a current research hotspot and challenge. ISFVs have recently attracted attention due to their high homology with pathogenic flaviviruses and unique inability to replicate in mammalian hosts. Multiple vaccine candidate strains constructed using ISFVs as scaffolds have demonstrated excellent safety and efficacy. This review summarizes the biological characteristics, host restriction factors, current applications in vaccine development, and challenges faced by ISFVs, providing a reference for future research on pathogenic flavivirus vaccines.

BACKGROUND:Pathogenic flaviviruses pose a serious threat to human health, and vaccines are an effective means of prevention and control. Although related vaccines have achieved significant progress, safety and efficacy limitations still exist, urgently requiring the development of novel vaccine platforms. The insect-specific flavivirus Chaoyang virus (CYV), with a structure similar to pathogenic flaviviruses and limited to insect cell replication, has potential as a safe vaccine vector. METHODS:To systematically evaluate CYV's potential as a universal flavivirus vaccine backbone and provide a vaccine candidate for type I Japanese encephalitis virus (JEV) prevention, this study constructed a chimeric JEV genotype I (GI) prME protein vaccine candidate CYV-JEV using CPER technology, systematically assessing its safety and immunoprotective effects. RESULTS:Using the CPER method, CYV-JEV was successfully rescued, showing efficient replication in mosquito cells but defective replication in mammalian cells. As a vaccine backbone, CYV did not induce inflammatory responses or immune cell subset imbalances in IFNAR-/- mice. CYV-JEV exhibited no pathogenicity in adult and suckling IFNAR-/- mice. Immunisation of IFNAR-/- mice with 106 FFU twice provided complete protection against lethal challenge (100%) and effectively reduced paralysis rates (62.5%). Single-cell sequencing further revealed extensive T- and B-cell activation in the immune spleen. CONCLUSIONS:The results demonstrate that the CYV-based CYV-JEV candidate vaccine demonstrates both safety and efficacy, representing a promising alternative to attenuated JEV vaccines, with CYV showing potential as a safe and effective universal flavivirus vaccine backbone.

Zika virus (ZIKV) is an emerging flavivirus that causes congenital syndromes including microcephaly and fetal demise in pregnant women. No commercial vaccines against ZIKV are currently available. We previously generated a chimeric ZIKV (ChinZIKV) based on the Chaoyang virus (CYV) by replacing the prME protein of CYV with that of a contemporary ZIKV strain GZ01. Herein, we evaluated this vaccine candidate in a mouse model and showed that ChinZIKV was totally safe in both adult and suckling immunodeficient mice. No viral RNA was detected in the serum of mice inoculated with ChinZIKV. All of the mice inoculated with ChinZIKV survived, while mice inoculated with ZIKV succumbed to infection in 8 days. A single dose of ChinZIKV partially protected mice against lethal ZIKV challenge. In contrast, all the control PBS-immunized mice succumbed to infection after ZIKV challenge. Our results warrant further development of ChinZIKV as a vaccine candidate in clinical trials.

The clinical treatment of hepatocellular carcinoma (HCC) is still a heavy burden worldwide. Intracellular microRNAs (miRNAs) commonly express abnormally in cancers, thus they are potential therapeutic targets for cancer treatment. miR-21 is upregulated in HCC whereas miR-122 is enriched in normal hepatocyte but downregulated in HCC. In our study, we first generated a reporter genetic switch compromising of miR-21 and miR-122 sponges as sensor, green fluorescent protein (GFP) as reporter gene and L7Ae:K-turn as regulatory element. The reporter expression was turned up in miR-21 enriched environment while turned down in miR-122 enriched environment, indicating that the reporter switch is able to respond distinctly to different miRNA environment. Furthermore, an AAT promoter, which is hepatocyte-specific, is applied to increase the specificity to hepatocyte. A killing switch with AAT promoter and an apoptosis-inducing element, Bax, in addition to miR-21 and miR-122 significantly inhibited cell viability in Huh-7 by 70 % and in HepG2 by 60 %. By contrast, cell viability was not affected in five non-HCC cells. Thus, we provide a novel feasible strategy to improve the safety of miRNA-based therapeutic agent to cancer.

Immunologic self-tolerance involves signals from co-inhibitory receptors. Several T cell co-inhibitors, including PD-1, are expressed upon activation, whereas CD5 and BTLA are expressed constitutively. The relationship between constitutively expressed co-inhibitors and when they are needed is unknown. Deletion of Btla demonstrated BTLA regulates CD5 expression. Loss of BTLA signals, but not signalling by its ligand, HVEM, leads to increased CD5 expression. Higher CD5 expression set during thymic selection is associated with increased self-recognition, suggesting that BTLA might be needed early to establish self-tolerance. We found that BTLA and PD-1 were needed post-thymic selection in recent thymic emigrants (RTE). RTE lacking BTLA caused a CD4 T cell and MHC class II dependent multi-organ autoimmune disease. Together, our findings identify a negative regulatory pathway between two constitutively expressed co-inhibitors, calibrating their expression. Expression of constitutive and induced co-inhibitory receptors is needed early to establish tolerance in the periphery for RTE.

Background: Uricase, or urate oxidase (Uox) is a key enzyme in uric acid (UA) metabolism and has been applied in clinical treatment of human hyperuricemia (HUA). However, the current clinically applied uricases, despite their potent urate-lowering capacity, tend to form anti-drug antibodies because of their immunogenicity, leading to increased risk of anaphylaxis, faster drug clearance and reduced or even complete loss of therapeutic effect, limiting their clinical application. In this study, we constructed engineered macrophages that stably expressed uricase, which might serve as a promising alternative to the direct injection of uricases. Materials and Methods: Engineered macrophages RAW264.7 cells were injected intravenously to treat hyperuricemic KM mice. Serum uric acid and bio-indicators for renal and hepatic functions were detected by an automatic biochemical analyzer; inflammatory cytokines were determined by ELISA; the livers and kidneys of the mice were sectioned for histological examination. Results: The uricase-expressing macrophages reduced UA levels from 300 ± 1.5 μmol/L to 101 ± 8.3 μmol/L in vitro. And in an HUA mouse model established by gavage with yeast extract, intravenous injection of the engineered macrophages could reduce the serum uric acid (sUA) of mice to normal level on the 14th day of modeling, with a decrease of 48.6%, and the urate-lowering effect was comparable to that of the first-line clinical drug allopurinol. In terms of safety, engineered macrophages did not cause liver or kidney dysfunction in mice, nor did they induce systemic immune response. Conclusions: Using macrophages as a chassis to deliver uricase might be a new, safe and effective strategy for the treatment and control of hyperuricemia.

Urate oxidase (Uox) plays a pivotal role in uric acid (UA) degradation, and it has been applied in controlling serum UA level in clinical treatment of hyperuricemia (HUA). However, because Uox is a heterogenous protein to the human body, the immune rejections typically occur after intravenous administration, which greatly hampers the application of Uox-based agents. In this study, we used Lactococcus lactis NZ9000, a food-grade bacterium, as a host to express exogenous Uox genes, to generate the Uox-expressing engineered strains to treat HUA. Aspergillus flavus-derived Uox (aUox) and the "resurrected" human-derived Uox (hUox) were cloned into vector and expressed in NZ9000, to generate engineered strains, respectively. The engineered NZ9000 strains were confirmed to express Uox and showed UA-lowering activity in a time-dependent manner in vitro. Next, in an HUA mice model established by oral gavage of yeast paste, the UA levels were increased by 85.4% and 106.2% at day 7 and day 14. By contrast, in mice fed with NZ9000-aUox, the UA levels were increased by 39.5% and 48.3% while in mice fed with NZ9000-hUox were increased by 57.0% and 82.9%, suggesting a UA-lowering activity of both engineered strains. Furthermore, compared with allopurinol, the first-line agent for HUA treatment, mice fed with NZ9000-aUox exhibited comparable liver safety but better kidney safety than allopurinol, indicating that the use of engineered NZ9000 strains not only alleviated kidney injury caused by HUA, but could also avoided the risk of kidney injury elicited by using allopurinol. Collectively, our study offers an effective and safe therapeutic approach for HUA long-term treatment and controlling.

In recent years, there have been frequent global outbreaks of viral epidemics such as Zika, COVID-19, and monkeypox, which have had a huge impact on human health and society and have also spurred innovation in virus engineering technology. The rise of synthetic virus genome technology has provided researchers with a new platform to accelerate vaccine and drug development. Although DNA synthesis technology has made significant progress, the current virus genome synthesis technology still requires the assembly of short oligonucleotides of around 60 bp into kb-level lengths when constructing long segments, a process in which the commonly used polymerase chain reaction assembly (PCA) technology has high error rates and is cumbersome to operate. This study optimized the error correction conditions after PCA assembly, increasing the accuracy of synthesizing 1 kb DNA fragments from 4.2 ± 2.1% before error correction to 31.3 ± 3.1% after two rounds of correction, an improvement of over 6 times. This study provides a more efficient operational process for synthesizing virus genomes from scratch, indicating greater potential for virus engineering in epidemic prevention and control and the field of biomedicine.

The plate counting method is a traditional and widely accepted technique for live cell counting, often employed for Bacillus enumeration and spore forming rate calculations. However, this method requires at least 12 h to generate results, making it unsuitable for real-time monitoring of bacterial growth status and spore transformation rate. Bacillus thuringiensis crystals, produced during sporulation, are widely used as microbial pesticides, with high demand for industrial scale production. Variations in cultivation conditions and harvest timing during large-scale pore production of Bacillus thuringiensis significantly affect spore forming rate, impacting crystallization yield. Nevertheless, there is a lack of real-time monitoring methods for spore conversion rate. Flow cytometry (FCM), a well-established technique for single-cell analysis in eukaryotic cells, has been successfully applied in bacterial detection in environmental and food samples. In this study, we introduced a rapid flow cytometry-based method for determining spore forming rate of Bacillus thuringiensis, with two nucleic acid dyes, SYTO24 and LDS751. The method enables dynamic monitoring of spore, vegetative cell, and viable but non-culturable/dead cell proportions during the whole cultivation process, and spore forming rate could be gained within 30 min. Data of spore forming rate by FCM method is consistent with that by plate counting method, offering a faster and more efficient approach for assessing sporulation status in industrial Bacillus thuringiensis microbial pesticide production.

Supplementary Figures 1-4 from Basal c-Jun NH2-terminal protein kinase activity is essential for survival and proliferation of T-cell acute lymphoblastic leukemia cells

The genus Flavivirus is a group of arthropod-borne single-stranded RNA viruses, which includes important human and animal pathogens such as Japanese encephalitis virus (JEV), Zika virus (ZIKV), Dengue virus (DENV), yellow fever virus (YFV), West Nile virus (WNV), and Tick-borne encephalitis virus (TBEV). Reverse genetics has been a useful tool for understanding biological properties and the pathogenesis of flaviviruses. However, the conventional construction of full-length infectious clones for flavivirus is time-consuming and difficult due to the toxicity of the flavivirus genome to E. coli. Herein, we applied a simple, rapid, and bacterium-free circular polymerase extension reaction (CPER) method to synthesize recombinant flaviviruses in vertebrate cells as well as insect cells. We started with the de novo synthesis of the JEV vaccine strain SA-14-14-2 in Vero cells using CPER, and then modified the CPER method to recover insect-specific flaviviruses (ISFs) in mosquito C6/36 cells. Chimeric Zika virus (ChinZIKV) based on the Chaoyang virus (CYV) backbone and the Culex flavivirus reporter virus expressing green fluorescent protein (CxFV-GFP) were subsequently rescued in C6/36 cells. CPER is a simple method for the rapid generation of flaviviruses and other potential RNA viruses. A CPER-based recovery system for flaviviruses of different host ranges was established, which would facilitate the development of countermeasures against flavivirus outbreaks in the future.

Botulinum neurotoxin serotype A (BoNT/A) can cause flaccid paralysis of muscles, an illness fatal to human, by entering neurons and blocking neurotransmitter release. The process was mediated by three receptors. A specific monoclonal antibody anti-D23, designated as ML419, targeting the ectodomain (D23) of fibroblast growth factor receptor 3 (FGFR3), one of the three receptors, was screened and capable of disturbing the recognition of BoNT/A and FGFR3. ML419 was screened from 14 stable positive hybridoma cell lines, and was subcloned, sequenced, and classified as IgG2a(κ) subclass. ML419 binds the D23 domain of FGFR3 with high affinity (KD∼0.26 nM), and prevents the BoNT/A from entering Neuro-2a cells effectively. In vivo data showed that, 200 μg of ML419 could completely protect all the mice against with 5 MLD50 BoNT/A, while 100 μg of ML419 could protected 60% of the mice. Collectively, our results indicated that ML419 served as a good candidate for further development of therapeutics for BoNT/A.

Asamitocins are maytansinoids produced by Actinosynnema pretiosum ssp. auranticum ATCC 31565 (A. pretiosum ATCC 31565), which have a structure similar to that of maytansine, therefore serving as a precursor of maytansine in the development of antibody-drug conjugates (ADCs). Currently, there are more than 20 known derivatives of ansamitocins, among which ansamitocin P-3 (AP-3) exhibits the highest antitumor activity. Despite its importance, the application of AP-3 is restricted by low yield, likely due to a substrate competition mechanism underlying the synthesis pathways of AP-3 and its byproducts. Given that N-demethylansamitocin P-3, the precursor of AP-3, is regulated by asm25 and asm10 to synthesize AGP-3 and AP-3, respectively, asm25 is predicted to be an inhibitory gene for AP-3 production. In this study, we inactivated asm25 in A. pretiosum ATCC 31565 by CRISPR-Cas9-guided gene editing. asm25 depletion resulted in a more than 2-fold increase in AP-3 yield. Surprisingly, the addition of isobutanol further improved AP-3 yield in the asm25 knockout strain by more than 6 times; in contrast, only a 1.53-fold increase was found in the WT strain under the parallel condition. Thus, we uncovered an unknown function of asm25 in AP-3 yield and identified asm25 as a promising target to enhance the large-scale industrial production of AP-3.

Intraepithelial T cells (IETs) are in close contact with intestinal epithelial cells and the underlying basement membrane, and they detect invasive pathogens. How intestinal epithelial cells and basement membrane influence IET survival and function, at steady state or after infection, is unclear. The herpes virus entry mediator (HVEM), a member of the TNF receptor superfamily, is constitutively expressed by intestinal epithelial cells and is important for protection from pathogenic bacteria. Here, we showed that at steady-state LIGHT, an HVEM ligand, binding to epithelial HVEM promoted the survival of small intestine IETs. RNA-seq and addition of HVEM ligands to epithelial organoids indicated that HVEM increased epithelial synthesis of basement membrane proteins, including collagen IV, which bound to β 1 integrins expressed by IETs. Therefore, we proposed that IET survival depended on β 1 integrin binding to collagen IV and showed that β 1 integrin–collagen IV interactions supported IET survival in vitro. Moreover, the absence of β 1 integrin expression by T lymphocytes decreased TCR αβ + IETs in vivo. Intravital microscopy showed that the patrolling movement of IETs was reduced without epithelial HVEM. As likely consequences of decreased number and movement, protective responses to Salmonella enterica were reduced in mice lacking either epithelial HVEM, HVEM ligands, or β 1 integrins. Therefore, IETs, at steady state and after infection, depended on HVEM expressed by epithelial cells for the synthesis of collagen IV by epithelial cells. Collagen IV engaged β 1 integrins on IETs that were important for their maintenance and for their protective function in mucosal immunity.

BACKGROUND:A large-scale outbreak of Zika virus (ZIKV) has occurred in Brazil and other South American countries, and has rapidly spread to 60 countries and regions worldwide since 2015, but no approved anti-ZIKV vaccines are available as of 2021.METHODS:We developed four types of anti-ZIKV DNA vaccine candidates: VPC-NS1, VPC-prME, VPC-prME-NS1, and VPC-EIII-NS1. They were developed against the structural proteins prM and E, and non-structural protein 1 (NS1) of ZIKV using the mammalian cell expression vector pcDNA3.1(+) as the backbone. For immunization, we intramuscularly injected mice with each vaccine candidate (n = 12 to 15 per group) on day 0 and day 14, with mice injected with phosphate-buffered saline (PBS) and pcDNA3.1(+) backbone vector as controls. On day 7, 21, and 35 after initial immunization, the effect of DNA vaccines was evaluated by ZIKV-specific humoral immunity determined by enzyme-linked immunosorbent assay (ELISA), ZIKV-specific T cell immunity determined by intracellular cytokine staining by flow cytometry and serum neutralization capacity determined by plaque reduction neutralization test (PRNT50) assay.RESULTS:The sequencing results showed that DNA vaccine vectors were successfully constructed. Western blotting and immunofluorescence results demonstrated the successful expression of immunogens carried by the DNA vaccines. On day 21 and 35 after the initial immunization, the levels of serum total immunoglobulin (Ig)G in all vaccine-given groups were slightly higher (approximately 1.5- to 2-fold) than those in the control groups. By contrast, ZIKV-specific IgG levels of all vaccine-given groups were significantly higher (approximately 10- to 1000- fold) than those of the control groups. The PRNT50 assay showed that the average serum dilution factors for neutralizing half ZIKV virions from vaccine-given groups were at least 32-fold (highest, 93-fold), while the sera from control group showed no protection. For cellular immunity, the proportions of CD11b+ myeloid cells, CD19+ B lymphocytes and CD3+ T lymphocytes in the mouse spleens as well as the percentages of CD4+ and CD8+ subsets of T cell were not changed 35 days after initial immunization. By contrast, the proportions of ZIKV-specific CD4+T cell and CD8+T cell in all vaccine-given groups were 2- to 10-folds and 2- to 30-fold than those in the control groups, respectively.CONCLUSION:All four DNA vaccines designed for the ZIKV induced neutralizing IgGs and cellular immune responses against ZIKV. Particularly, VPC-EIII-NS1 induced high level of humoral response comparable to the vaccine candidate containing prM, E and NS1 polyprotein, suggesting a potent reduced ADE effect and reserved neutralizing activity. Our findings may provide guidance for improving safety of anti-ZIKV vaccines in the future.

Fulminant hepatitis (FH) is a life-threatening disease with partially understood pathogenesis.It has been demonstrated that myeloid-derived suppressor cells (MDSCs) are recruited into the liver during this process, and their augmented accumulation by various strategies protects against liver injury.However, the underlying mechanism(s) remain elusive.Receptor for activated C kinase 1 (RACK1), a multi-functional scaffold protein, is highly expressed in normal liver and has been implicated in liver physiology and diseases, but the in vivo role of hepatic RACK1 in FH remains unknown.Methods: Survival curves and liver damage were monitored to investigate the in vivo role of hepatic RACK1 in FH.The liver microenvironment was explored by microarray-based transcriptome analysis, flow cytometry, immunoblotting, and immunohistochemistry. MDSCs were identified with phenotypic and functional characteristics.Functional antibodies were used to target MDSCs.Co-culture techniques were used to study the underlying mechanism(s) of protection.The interaction of RACK1 with histone deacetylase 1 (HDAC1) and the consequent effects on HDAC1 ubiquitination were analyzed.Ectopic expression of HDAC1 with recombinant adeno-associated virus serotype 8 was conducted to confirm the role of HDAC1 in the protective effects of hepatic RACK1 deficiency against FH.Post-translational modifications of RACK1 were also investigated during the induction of FH.Results: Liver-specific RACK1 deficiency rendered mice resistant to FH. RACK1-deficient livers exhibited high basal levels of chemokine (C-X-C motif) ligand 1 (CXCL1) and S100 calcium-binding protein A9 (S100A9), associated with MDSC accumulation under steady-state conditions.Targeting MDSCs with an antibody against either Gr1 or DR5 abrogated the protective effects of liver-specific RACK1 deficiency.Accumulated MDSCs inhibited inflammatory cytokine production from macrophages and enhanced IκB kinase (IKK)/NF-κB pathway activation in hepatocytes.Further investigation revealed that RACK1 maintained HDAC1 protein level in hepatocytes by direct binding, thereby controlling histone H3K9 and H3K27 acetylation at the Cxcl1 and S100a9 promoters.Ectopic expression of HDAC1 in livers with RACK1 deficiency partially reversed the augmented Cxcl1/S100a9 → MDSCs → IKK/NF-κB axis.During FH induction, RACK1 was phosphorylated at serine 110, enhancing its binding to ubiquitin-conjugating enzyme E2T and promoting its ubiquitination and degradation.Conclusion: Liver-specific RACK1 deficiency protects against FH through accelerated HDAC1 degradation and the consequent CXCL1/S100A9 upregulation and MDSC accumulation.

Objective To establish a novel hepatocyte injury model induced by lipopolysaccharide/D-galactosamine (LPS/D-GalN) in vitro. Methods Freshly isolated mouse primary hepatocytes were cultured in vitro and treated with different doses of tumor necrosis factor-α (TNF-α) and 5 mg/mL of D-GalN. The supernatants from hepatocyte culture were detected for alanine aminotransferase (ALT) activity by chemiluminescence assay. Bone marrow-derived macrophages (BMDMs) were stimulated with 1 μg/mL of LPS and the level of TNF-α in supernatants were detected by ELISA. Primary hepatocytes were treated with the BMDM supernatants combined with 5 mg/mL D-GalN or 50 ng/mL actinomycin D (ActD) for 24 hours. The level of ALT from hepatocyte supernatant was detected and morphology of hepatocytes was observed with microscopy. BMDMs and hepatocytes were co-cultured and treated with 1 μg/mL of LPS combined with D-GalN or ActD for 24 hours. Hepatocyte injury was reflected by the ALT activity and hepatocyte morphology. Results The ALT activity was significantly increased in the supernatants of hepatocytes treated with TNF-α and D-GalN, indicating the obvious hepatocyte injury. Co-treatment with LPS-primed BMDM supernatants and D-GalN or ActD could cause hepatocyte injury, as reflected by markedly increased ALT activity and the deformed and cracked hepatocytes. In the context of co-culture of BMDM and hepatocytes, treatment with LPS and D-GalN led to obvious hepatocyte injury as expected. LPS combined with ActD could not cause hepatocyte injury, since the BMDMs started to die earlier than they could secret TNF-α to destruct hepatocytes. Hepatocytes with normal morphology and deformed BMDMs were observed. Conclusion LPS/D-GalN can be used to induce hepatocyte injury in vitro. D-GalN, rather than ActD, should be used as a transcriptional inhibitor when the TNF-α -induced hepatocyte injury is evaluated in a co-culture system of BMDMs and hepatocytes.

The dynamics, duration, and nature of immunity produced during SARS-CoV-2 infection are still unclear. Here, we longitudinally measured virus-neutralising antibody, specific antibodies against the spike (S) protein, receptor-binding domain (RBD), and the nucleoprotein (N) of SARS-CoV-2, as well as T cell responses, in 25 SARS-CoV-2-infected patients up to 121 days post-symptom onset (PSO). All patients seroconvert for IgG against N, S, or RBD, as well as IgM against RBD, and produce neutralising antibodies (NAb) by 14 days PSO, with the peak levels attained by 15–30 days PSO. Anti-SARS-CoV-2 IgG and NAb remain detectable and relatively stable 3–4 months PSO, whereas IgM antibody rapidly decay. Approximately 65% of patients have detectable SARS-CoV-2-specific CD4 + or CD8 + T cell responses 3–4 months PSO. Our results thus provide critical evidence that IgG, NAb, and T cell responses persist in the majority of patients for at least 3–4 months after infection.