Osr2 functions as a biomechanical checkpoint to aggravate CD8+ T cell exhaustion in tumor.

Alterations in extracellular matrix (ECM) architecture and stiffness represent hallmarks of cancer. Whether the biomechanical property of ECM impacts the functionality of tumor-reactive CD8+ T cells remains largely unknown. Here, we reveal that the transcription factor (TF) Osr2 integrates biomechanical signaling and facilitates the terminal exhaustion of tumor-reactive CD8+ T cells. Osr2 expression is selectively induced in the terminally exhausted tumor-specific CD8+ T cell subset by coupled T cell receptor (TCR) signaling and biomechanical stress mediated by the Piezo1/calcium/CREB axis. Consistently, depletion of Osr2 alleviates the exhaustion of tumor-specific CD8+ T cells or CAR-T cells, whereas forced Osr2 expression aggravates their exhaustion in solid tumor models. Mechanistically, Osr2 recruits HDAC3 to rewire the epigenetic program for suppressing cytotoxic gene expression and promoting CD8+ T cell exhaustion. Thus, our results unravel Osr2 functions as a biomechanical checkpoint to exacerbate CD8+ T cell exhaustion and could be targeted to potentiate cancer immunotherapy.

Swine pseudorabies virus attenuated vaccine reprograms the kidney cancer tumor microenvironment and synergizes with PD-1 blockade.

The global incidence rate of kidney cancer (KC) has been steadily increasing over the past 30 years. With the aging global population, kidney cancer has become an escalating concern that necessitates vigilant surveillance. Nowadays, surgical intervention remains the optimal therapeutic approach for kidney cancer, while the availability of efficacious treatments for advanced tumors remains limited. Oncolytic viruses, an emerging form of immunotherapy, have demonstrated encouraging anti-neoplastic properties and are progressively garnering public acceptance. However, research on oncolytic viruses in kidney cancer is relatively limited. Furthermore, given the high complexity and heterogeneity of kidney cancer, it is crucial to identify an optimal oncolytic virus agent that is better suited for its treatment. The present study investigates the oncolytic activity of the Pseudorabies virus live attenuated vaccine (PRV-LAV) against KC. The findings clearly demonstrate that PRV-LAV exhibits robust oncolytic activity targeting KC cell lines. Furthermore, the therapeutic efficacy of PRV-LAV was confirmed in both a subcutaneous tumor-bearing nude mouse model and a syngeneic mouse model of KC. Combined RNA-seq analysis and flow cytometry revealed that PRV-LAV treatment substantially enhances the infiltration of a diverse range of lymphocytes, including T cells, B cells, macrophages, and NK cells. Additionally, PRV-LAV treatment enhances T cell activation and exerts antitumor effects. Importantly, the combination of PRV-LAV with anti-PD-1 antibodies, an approved drug for KC treatment, synergistically enhances the efficacy against KC. Overall, the discovery of PRV-LAV as an effective oncolytic virus holds significant importance for improving the treatment efficacy and survival rates of KC patients.

Human amniotic mesenchymal stromal cell-derived exosomes promote neuronal function by inhibiting excessive apoptosis in a hypoxia/ischemia-induced cerebral palsy model: A preclinical study.

BACKGROUND: Cerebral palsy (CP) is a condition resulting from perinatal brain injury and can lead to physical disabilities. Exosomes derived from human amniotic mesenchymal stromal cells (hAMSC-Exos) hold promise as potential therapeutic options. OBJECTIVE: This study aimed to investigate the impact of hAMSC-Exos on neuronal cells and their role in regulating apoptosis both in vitro and in vivo. METHODS: hAMSC-Exos were isolated via ultracentrifugation and characterized via transmission electron microscopy, particle size analysis, and flow cytometry. In vitro, neuronal damage was induced by lipopolysaccharide (LPS). CP rat models were established via left common carotid artery ligation. Apoptosis levels in cells and CP rats were assessed using flow cytometry, quantitative reverse transcription polymerase chain reaction (RT-qPCR), Western blotting, and TUNEL analysis. RESULTS: The results demonstrated successful isolation of hAMSC-Exos via ultracentrifugation, as the isolated cells were positive for CD9 (79.7%) and CD63 (80.2%). Treatment with hAMSC-Exos significantly mitigated the reduction in cell viability induced by LPS. Flow cytometry revealed that LPS-induced damage promoted apoptosis, but this effect was attenuated by treatment with hAMSC-Exos. Additionally, the expression of caspase-3 and caspase-9 and the Bcl-2/Bax ratio indicated that excessive apoptosis could be attenuated by treatment with hAMSC-Exos. Furthermore, tail vein injection of hAMSC-Exos improved the neurobehavioral function of CP rats. Histological analysis via HE and TUNEL staining showed that apoptosis-related damage was attenuated following hAMSC-Exo treatment. CONCLUSIONS: In conclusion, hAMSC-Exos effectively promote neuronal cell survival by regulating apoptosis, indicating their potential as a promising therapeutic option for CP that merits further investigation.

Dual inhibition of CYP17A1 and HDAC6 by abiraterone-installed hydroxamic acid overcomes temozolomide resistance in glioblastoma through inducing DNA damage and oxidative stress.

Glioblastoma (GBM) is a highly aggressive and treatment-resistant brain tumor, necessitating novel therapeutic strategies. In this study, we present a mechanistic breakthrough by designing and evaluating a series of abiraterone-installed hydroxamic acids as potential dual inhibitors of CYP17A1 and HDAC6 for GBM treatment. We established the correlation of CYP17A1/HDAC6 overexpression with tumor recurrence and temozolomide resistance in GBM patients. Compound 12, a dual inhibitor, demonstrated significant anti-GBM activity in vitro, particularly against TMZ-resistant cell lines. Mechanistically, compound 12 induced apoptosis, suppressed recurrence-associated genes, induced oxidative stress and initiated DNA damage response. Furthermore, molecular modeling studies confirmed its potent inhibitory activity against CYP17A1 and HDAC6. In vivo studies revealed that compound 12 effectively suppressed tumor growth in xenograft and orthotopic mouse models without inducing significant adverse effects. These findings highlight the potential of dual CYP17A1 and HDAC6 inhibition as a promising strategy for overcoming treatment resistance in GBM and offer new hope for improved therapeutic outcomes.

Non-overlapping epitopes on the gHgL-gp42 complex for the rational design of a triple-antibody cocktail against EBV infection.

Epstein-Barr virus (EBV) is closely associated with cancer, multiple sclerosis, and post-acute coronavirus disease 2019 (COVID-19) sequelae. There are currently no approved therapeutics or vaccines against EBV. It is noteworthy that combining multiple EBV glycoproteins can elicit potent neutralizing antibodies (nAbs) against viral infection, suggesting possible synergistic effects. Here, we characterize three nAbs (anti-gp42 5E3, anti-gHgL 6H2, and anti-gHgL 10E4) targeting different glycoproteins of the gHgL-gp42 complex. Two antibody cocktails synergistically neutralize infection in B cells (5E3+6H2+10E4) and epithelial cells (6H2+10E4) in vitro. Moreover, 5E3 alone and the 5E3+6H2+10E4 cocktail confer potent in vivo protection against lethal EBV challenge in humanized mice. The cryo-EM structure of a heptatomic gHgL-gp42 immune complex reveals non-overlapping epitopes of 5E3, 6H2, and 10E4 on the gHgL-gp42 complex. Structural and functional analyses highlight different neutralization mechanisms for each of the three nAbs. In summary, our results provide insight for the rational design of therapeutics or vaccines against EBV infection.

Listeria monocytogenes: a promising vector for tumor immunotherapy.

Cancer receives enduring international attention due to its extremely high morbidity and mortality. Immunotherapy, which is generally expected to overcome the limits of traditional treatments, serves as a promising direction for patients with recurrent or metastatic malignancies. Bacteria-based vectors such as Listeria monocytogenes take advantage of their unique characteristics, including preferential infection of host antigen presenting cells, intracellular growth within immune cells, and intercellular dissemination, to further improve the efficacy and minimize off-target effects of tailed immune treatments. Listeria monocytogenes can reshape the tumor microenvironment to bolster the anti-tumor effects both through the enhancement of T cells activity and a decrease in the frequency and population of immunosuppressive cells. Modified Listeria monocytogenes has been employed as a tool to elicit immune responses against different tumor cells. Currently, Listeria monocytogenes vaccine alone is insufficient to treat all patients effectively, which can be addressed if combined with other treatments, such as immune checkpoint inhibitors, reactivated adoptive cell therapy, and radiotherapy. This review summarizes the recent advances in the molecular mechanisms underlying the involvement of Listeria monocytogenes vaccine in anti-tumor immunity, and discusses the most concerned issues for future research.

Mesenchymal Stem Cells Overexpressing FGF21 Preserve Blood-Brain Barrier Integrity in Experimental Ischemic Stroke.

Blood-brain barrier (BBB) disruption is a prominent pathophysiological mechanism in stroke. Transplantation of mesenchymal stem cells (MSCs) preserves BBB integrity following ischemic stroke. Fibroblast growth factor 21 (FGF21) has been shown to be a potent neuroprotective agent that reduces neuroinflammation and protects against BBB leakage. In this study, we assessed the effects of transplantation of MSCs overexpressing FGF21 (MSCs-FGF21) on ischemia-induced neurological deficits and BBB breakdown. MSCs-FGF21 was injected into the rat brain via the intracerebroventricular route 24 h after middle cerebral artery occlusion (MCAO) surgery. The behavioral performance was assessed using modified neurological severity scores and Y-maze tests. BBB disruption was measured using Evans blue staining, IgG extravasation, and brain water content. The levels of tight junction proteins, aquaporin 4, and neuroinflammatory markers were analyzed by western blotting and immunohistochemistry. The activity of matrix metalloproteinase-9 (MMP-9) was determined using gelatin zymography. At day-5 after MCAO surgery, intraventricular injection of MSCs-FGF21 was found to significantly mitigate the neurological deficits and BBB disruption. The MCAO-induced loss of tight junction proteins, including ZO-1, occludin, and claudin-5, and upregulation of the edema inducer, aquaporin 4, were also remarkably inhibited. In addition, brain infarct volume, pro-inflammatory protein expression, and MMP-9 activation were effectively suppressed. These MCAO-induced changes were only marginally improved by treatment with MSCs-mCherry, which did not overexpress FGF21. Overexpression of FGF21 dramatically improved the therapeutic efficacy of MSCs in treating ischemic stroke. Given its multiple benefits and long therapeutic window, MSC-FGF21 therapy may be a promising treatment strategy for ischemic stroke.

Neutralizing antibodies against EBV gp42 show potent in vivo protection and define novel epitopes.

Epstein-Barr virus (EBV) is the first reported human oncogenic virus and infects more than 95% of the human population worldwide. EBV latent infection in B lymphocytes is essential for viral persistence. Glycoprotein gp42 is an indispensable member of the triggering complex for EBV entry into B cells. The C-type lectin domain (CTLD) of gp42 plays a key role in receptor binding and is the major target of neutralizing antibodies. Here, we isolated two rabbit antibodies, 1A7 and 6G7, targeting gp42 CTLD with potent neutralizing activity against B cell infection. Antibody 6G7 efficiently protects humanized mice from lethal EBV challenge and EBV-induced lymphoma. Neutralizing epitopes targeted by antibodies 1A7 and 6G7 are distinct and novel. Antibody 6G7 blocks gp42 binding to B cell surface and both 1A7 and 6G7 inhibit membrane fusion with B cells. Furthermore, 1A7- and 6G7-like antibodies in immunized sera are major contributors to B cell neutralization. This study demonstrates that anti-gp42 neutralizing antibodies are effective in inhibiting EBV infection and sheds light on the design of gp42-based vaccines and therapeutics.

Therapeutic Effect of Human Adipocyte-derived Stem Cell-derived Exosomes on a Transgenic Mouse Model of Parkinson's Disease.

BACKGROUND/AIM: Stem cell therapy and regenerative medicine are promising for treating Parkinson's disease (PD) not only for the potential for cell replacement but also for the paracrine effect of stem cell secretion, especially proteins and nucleotide-enriched exosomes. This study investigated the neuroprotective effect of exosomes secreted from human adipocyte-derived stem cells (hADSCs) on PD. MATERIALS AND METHODS: hADSCs were isolated from the visceral fat tissue of individuals without PD who underwent bariatric surgery and were validated using surface markers and differentiation ability. Exosomes were isolated from the culture medium of hADSCs through serial ultracentrifugation and validated. Condensed exosomes were administered intravenously to 12-week-old MitoPark mice, transgenic parkinsonism mouse model with conditional knockout of mitochondrial transcription factor A in dopaminergic neurons, monthly for 3 months. Motor function, gait, and memory were assessed monthly, and immunohistochemical analysis of neuronal and inflammatory markers was performed at the end of the experiments. RESULTS: The hADSC-derived exosome-treated mice exhibited better motor function in beam walking and gait analyses than did the untreated mice. In the novel object recognition tests, the exosome-treated mice retained better memory function. Immunohistochemical analysis revealed that although exosome treatment did not prevent the loss of dopaminergic neurons in the substantia nigra of mice, it down-regulated microglial activation and neuroinflammation in the midbrain. CONCLUSION: hADSC-derived exosomes were neuroprotective in this in vivo mouse model of PD, likely because of their anti-inflammatory effect. Use of hADSC-derived exosomes may offer several beneficial effects in stem cell therapy. Since they can also be produced at an industrial level, they are a promising treatment option for PD and other neurodegenerative diseases.

[Corrigendum] Crocetin induces apoptosis of BGC­823 human gastric cancer cells.

Following the publication of the above article, an interested reader drew to the authors' attention that Fig. 2 (showing morphological characteristics of cultured BGC­823 cells as visualized by microscopic analysis) and Fig. 3 (showing crocetin­induced apoptosis of the BGC­823 cells) on p. 523 appeared to feature panels containing overlapping data. The authors re­examined their original data, and realized that inadvertent errors were made during the compilation of these figures; specifically, the data shown in Fig. 2C (for the the 5­µM docetaxel group) and Fig. 3D (for the DMSO group) were selected incorrectly. The corrected versions of Figs. 2 and 3 are shown below and on the next page, now featuring the correct data for Figs. 2C and 3D. All the authors agree with the publication of this corrigendum, and are grateful of the Editor of Molecular Medicine Reports for granting them the opportunity to publish this. Furthermore, they regret that these errors were introduced into the paper, even though they did not substantially alter any of the major conclusions reported in the paper, and apologize to the readership for any inconvenience caused. [Molecular Medicine Reports 9: 521­526, 2014; DOI: 10.3892/mmr.2013.1851].

MCM6 promotes intrahepatic cholangiocarcinoma progression by upregulating E2F1 and enhancing epithelial-mesenchymal transition.

Minichromosome maintenance complex component 6 (MCM6), a member of the MCM family, plays a pivotal role in DNA replication initiation and genome duplication of proliferating cells. MCM6 is upregulated in multiple malignancies and is considered a novel diagnostic biomarker. However, the functional contributions and prognostic value of MCM6 in intrahepatic cholangiocarcinoma (ICC) remain unexplored. In this study, we investigated the molecular function of MCM6 in ICC. Data from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO, GSE107943) indicated an upregulation of MCM6 in tumor tissues. Immunohistochemical analysis performed on 115 cases of ICC samples confirmed the upregulation of MCM6 and further suggested that a high level of MCM6 expression predicted shorter overall and disease-free survival in ICC patients. Functional studies suggested that MCM6 knockdown significantly suppressed cell viability, blocked cell cycle progression and inhibited metastasis, while the enhancement of MCM6 expression promoted the proliferation and migration of ICC cells both in vitro and in vivo. Mechanistically, Gene Set Enrichment Analysis (GSEA) suggested that the epithelial-mesenchymal transition (EMT) and E2F1-correlated genes were enriched in ICC tissues with high MCM6 expression. Further verification indicated that MCM6 promoted the EMT of ICC cells via upregulating E2F1. In addition, E2F1 knockdown partially blocked the pro-malignant effects of MCM6 overexpression. In summary, MCM6 was found to be a novel prognostic and predictive marker for ICC. MCM6 promoted ICC progression via activation of E2F1-mediated EMT.

[Retracted] SIRT1 promotes tumorigenesis of hepatocellular carcinoma through PI3K/PTEN/AKT signaling.

Following the publication of this paper, it was drawn to the Editor's attention by a concerned reader that the control ß­actin western blots shown in Figs. 1B and 6 were strikingly similar to data that had already appeared in a different form in the following publication: Lei Y, Liu H, Yang Y, Wang X, Ren N, Li B, Liu S, Cheng J, Fu X and Zhang J: Interaction of LHBs with C53 promotes hepatocyte mitotic entry: A novel mechanism for HBV­induced hepatocellular carcinoma. Oncol Rep 29: 151­159, 2012. Owing to the fact that the contentious data in the above article had already been published prior to its submission to Oncology Reports, the Editor has decided that this paper should be retracted from the Journal. After having been in contact with the authors, they accepted the decision to retract the paper. The Editor apologizes to the readership for any inconvenience caused. [Oncology Reports 28: 311­318, 2012; DOI: 10.3892/or.2012.1788].

Serine/threonine kinase TBK1 promotes cholangiocarcinoma progression via direct regulation of ß-catenin.

Cholangiocarcinoma (CCA) is a highly heterogeneous and metastatic malignancy with a poor prognosis even after curative hepatectomy. Studies exploring its pathogenesis and identifying effective therapeutic targets are urgently needed. In this study, we found that TANK-binding kinase 1 (TBK1), a serine/threonine-protein kinase, showed a dynamic increase during the different stages of murine spontaneous CCA carcinogenesis (hyperplasia, dysplasia, and CCA). TBK1 was upregulated in human tissues, including intrahepatic (n = 182) and extrahepatic (n = 40) CCA tissues, compared with nontumor tissues, and the elevated expression of TBK1 was positively correlated with larger tumour diameter, lymph node metastasis, and advanced TNM stage. Functional studies indicated that TBK1 promoted CCA growth and metastasis both in vitro and in vivo. TBK1 directly interacts with ß-catenin, promoting its phosphorylation at the S552 site and its nuclear translocation, which further activates EMT-related transcriptional reprogramming. GSK-8612, a TBK1 inhibitor or a kinase-inactivating mutation, effectively suppresses the above processes. In addition, we found that low-density lipoprotein receptor (LDLR), which mediates the endocytosis of cholesterol, was upregulated in CCA. Therefore, we designed a cholesterol-conjugated DNA/RNA heteroduplex oligonucleotide targeting TBK1 (Cho-TBK1-HDO), which could accumulate in CCA cells via LDLR, reduce the TBK1 mRNA level and inhibit intrahepatic metastasis of CCA. Besides, in the experimental group of 182 ICC patients, high TBK1 expression combined with high nuclear ß-catenin expression predicted a worse prognosis. In summary, TBK1 might serve as a potential prognostic biomarker and therapeutic target for patients with CCA.

Screening and identification of potential key biomarkers for glucocorticoid-induced osteonecrosis of the femoral head.

BACKGROUND: Glucocorticoid-induced osteonecrosis of the femoral head (GIONFH) is a common disease in osteoarticular surgery, with a high disability rate, which brings great physical and mental pain and economic burden to patients. Its specific pathogenesis has not been fully demonstrated, and there is a lack of recognized effective biomarkers for earlier detection and prompt treatment. This has become an urgent clinical problem for orthopedic scholars. MATERIALS AND METHODS: We downloaded the gene expression profile dataset GSE123568 from the Gene Expression Omnibus database, used STRING and Cytoscape to carry out module analysis and built a gene interaction network. The four core genes most related to GIONFH in this network were ultimately found out by precise analysis and animal experiment were then conducted for verification. In this verification process, thirty-six New Zealand white rabbits were randomly divided into blank control group, model group and drug group. Except for the blank control group, the animal model of GIONFH was established by lipopolysaccharide and methylprednisolone, while the drug group was given the lipid-lowering drugs for intervention as planned. The rabbits were taken for magnetic resonance imaging at different stages, and their femoral head specimens were taken for pathological examination, then the expression of target genes in the femoral head specimens of corresponding groups was detected. Validation methods included RT-PCR and pathological examination. RESULTS: A total of 679 differential genes were selected at first, including 276 up-regulated genes and 403 down-regulated genes. Finally, four genes with the highest degree of correlation were screened. Animal experiment results showed that ASXL1 and BNIP3L were in low expression, while FCGR2A and TYROBP were highly expressed. CONCLUSION: Through animal experiments, it was confirmed that ASXL1, BNIP3L, FCGR2A and TYROBP screened from the comparative analysis of multiple genes in the database were closely related to GIONFH, which is important for early diagnosis of Glucocorticoid-induced osteonecrosis of the femoral head.

Rifaximin Modifies Gut Microbiota and Attenuates Inflammation in Parkinson's Disease: Preclinical and Clinical Studies.

Patients with Parkinson's disease (PD) exhibit distinct gut microbiota, which may promote gut-derived inflammation. Rifaximin is a nonabsorbable antibiotic that can modify gut microbiota. The present study investigated the effect of rifaximin on gut microbiota and inflammation status in PD. The study examined the effect of long-term rifaximin treatment on in vivo transgenic PD mice (MitoPark) and short-term rifaximin treatment on patients with PD. Rifaximin treatment caused a significant change in gut microbiota in the transgenic PD mice; in particular, it reduced the relative abundance of Prevotellaceae UCG-001 and increased the relative abundance of Bacteroides, Muribaculum, and Lachnospiraceae UCG-001. Rifaximin treatment attenuated serum interleukin-1ß, interleukin-6 and tumor necrosis factor-α, claudin-5 and occludin, which indicated the reduction of systemic inflammation and the protection of the blood-brain barrier integrity. The rifaximin-treated MitoPark mice exhibited better motor and memory performance than did the control mice, with lower microglial activation and increased neuronal survival in the hippocampus. In the patients with PD, 7-day rifaximin treatment caused an increase in the relative abundance of Flavonifractor 6 months after treatment, and the change in plasma proinflammatory cytokine levels was negatively associated with the baseline plasma interleukin-1α level. In conclusion, the present study demonstrated that rifaximin exerted a neuroprotective effect on the transgenic PD mice by modulating gut microbiota. We observed that patients with higher baseline inflammation possibly benefited from rifaximin treatment. With consideration for the tolerability and safety of rifaximin, randomized controlled trials should investigate the disease-modification effect of long-term treatment on select patients with PD.

LncRNA CRNDE is involved in radiation resistance in hepatocellular carcinoma via modulating the SP1/PDK1 axis.

Hepatocellular carcinoma (HCC) is defined as a universal malignancy while radiation therapy is the effective treatment for it. This study validated the mechanism of long non-coding RNA (lncRNA) colorectal neoplasia differentially expressed gene (CRNDE) in radiation resistance in HCC. LncRNA CRNDE upregulation was detected in HCC cells. The radiation-resistant cell strains Huh7R and SNU-387R were established. After silencing lncRNA CRNDE, the cell colony formation ability, cell activity, apoptosis, cell cycles, and γ-H2AX positive rate in Huh7R and SNU-387R were detected. Silencing lncRNA CRNDE decreased the cell activity, colony formation ability, and cell number in the G2 phase and facilitated DNA damage and apoptosis. The binding relations of specificity protein 1 (SP1) with lncRNA CRNDE and 3-phosphoinositide dependent protein kinase 1 (PDK1) were verified. LncRNA CRNDE regulated PDK1 transcription by binding to transcription factor SP1. PDK1 overexpression partially reversed the inhibition of silencing lncRNA CRNDE on radiation resistance in HCC cells. The transplanted tumor mouse model was established and showed that silencing lncRNA CRNDE decreased tumor volume and weight and Ki67-positive cells in HCC mice in vivo. Collectively, lncRNA CRNDE was upregulated in HCC cells and promoted PDK1 transcription by binding to SP1, thus enhancing radiation resistance in HCC cells.

Glycoprotein B Antibodies Completely Neutralize EBV Infection of B Cells.

The Epstein-Barr virus (EBV) is the first reported oncogenic herpesvirus that establishes persistent infection in B lymphocytes in 95% of adults worldwide. Glycoprotein B (gB) plays a predominant role in the fusion of the viral envelope with the host cell membrane. Hence, it is of great significance to isolate gB-specific fusion-inhibiting neutralizing antibodies (NAbs). AMMO5 is the only gB NAb but fails to antagonize B-cell infection. It is essential to isolate potent NAbs that can completely block EBV infection of B cells. Using hybridoma technology and neutralization assay, we isolate two gB NAbs 8A9 and 8C12 that are capable of completely neutralizing B-cell infection in vitro. In addition, 8A9 shows cross-reactivity with rhesus lymphocryptovirus (rhLCV) gB. Competitive binding experiments demonstrate that 8A9 and 8C12 recognize novel epitopes that are different from the AMMO5 epitope. The epitopes of 8A9 and 8C12 are mapped to gB D-II, and the AMMO5 epitope is located precisely at gB aa 410-419. We find that 8A9 and 8C12 significantly inhibit gB-derived membrane fusion using a virus-free fusion assay. In summary, this study identifies two gB-specific NAbs that potently block EBV infection of B cells. Our work highlights the importance of gB D-II as a predominant neutralizing epitope, and aids in the rational design of therapeutics or vaccines based on gB.

3,6'- and 1,6'-Dithiopomalidomide Mitigate Ischemic Stroke in Rats and Blunt Inflammation.

(1) Background: An important concomitant of stroke is neuroinflammation. Pomalidomide, a clinically available immunomodulatory imide drug (IMiD) used in cancer therapy, lowers TNF-α generation and thus has potent anti-inflammatory actions. Well-tolerated analogs may provide a stroke treatment and allow evaluation of the role of neuroinflammation in the ischemic brain. (2) Methods: Two novel pomalidomide derivatives, 3,6'-dithiopomalidomide (3,6'-DP) and 1,6'-dithiopomalidomide (1,6'-DP), were evaluated alongside pomalidomide in a rat middle cerebral artery occlusion (MCAo) stroke model, and their anti-inflammatory actions were characterized. (3) Results: Post-MCAo administration of all drugs lowered pro-inflammatory TNF-α and IL1-ß levels, and reduced stroke-induced postural asymmetry and infarct size. Whereas 3,6'- and 1,6'-DP, like pomalidomide, potently bound to cereblon in cellular studies, 3,6'-DP did not lower Ikaros, Aiolos or SALL4 levels-critical intermediates mediating the anticancer/teratogenic actions of pomalidomide and IMiDs. 3,6'-DP and 1,6'-DP lacked activity in mammalian chromosome aberration, AMES and hERG channel assays -critical FDA regulatory tests. Finally, 3,6'- and 1,6'-DP mitigated inflammation across rat primary dopaminergic neuron and microglia mixed cultures challenged with α-synuclein and mouse LPS-challenged RAW 264.7 cells. (4) Conclusion: Neuroinflammation mediated via TNF-α plays a key role in stroke outcome, and 3,6'-DP and 1,6'-DP may prove valuable as stroke therapies and thus warrant further preclinical development.

A Neutralizing Antibody Targeting gH Provides Potent Protection against EBV Challenge In Vivo.

Epstein-Barr virus (EBV) is an oncogenic herpesvirus that is associated with 200,000 new cases of cancer and 140,000 deaths annually. To date, there are no available vaccines or therapeutics for clinical usage. Recently, the viral heterodimer glycoprotein gH/gL has become a promising target for the development of prophylactic vaccines against EBV. Here, we developed the anti-gH antibody 6H2 and its chimeric version C6H2, which had full neutralizing activity in epithelial cells and partial neutralizing activity in B cells. C6H2 exhibited potent protection against lethal EBV challenge in a humanized mouse model. The cryo-electron microscopy (cryo-EM) structure further revealed that 6H2 recognized a previously unidentified epitope on gH/gL D-IV that is critical for viral attachment and subsequent membrane fusion with epithelial cells. Our results suggest that C6H2 is a promising candidate in the prevention of EBV-induced lymphoproliferative diseases (LPDs) and may inform the design of an EBV vaccine. IMPORTANCE Epstein-Barr virus (EBV) is a ubiquitous gammaherpesvirus that establishes lifelong persistence and is related to multiple diseases, including cancers. Neutralizing antibodies (NAbs) have proven to be highly effective in preventing EBV infection and subsequent diseases. Here, we developed an anti-EBV-gH NAb, 6H2, which blocked EBV infection in vitro and in vivo. This 6H2 neutralizing epitope should be helpful to understand EBV infection mechanisms and guide the development of vaccines and therapeutics against EBV infection.

Antibody Generation and Immunogenicity Analysis of EBV gp42 N-Terminal Region.

Epstein-Barr virus (EBV) is the first reported oncogenic virus and infects more than 90% of adults worldwide. EBV can establish a latent infection in B lymphocytes which is essential for persistence and transmission. Glycoprotein gp42 is an indispensable member of the triggering complex for EBV entry into a B cell. The N-terminal region of gp42 plays a key role in binding to gH/gL and triggering subsequent membrane fusion. However, no antibody has been reported to recognize this region and the immunogenicity of gp42 N-domain remains unknown. In the present study, we have generated a panel of nine mAbs against the gp42 N-terminal region (six mAbs to gp42-44-61aa and three mAbs to gp42-67-81aa). These mAbs show excellent binding activity and recognize different key residues locating on the gp42 N-domain. Among the nine mAbs, 4H7, 4H8 and 11G10 cross-react with rhLCV-gp42 while other mAbs specifically recognize EBV-gp42. Our newly obtained mAbs provide a useful tool for investigating the gp42 function and viral infection mechanism of γ-Herpesvirus. Furthermore, we assess the immunogenicity of the gp42 N-terminal region using the HBc149 particle as a carrier protein. The chimeric VLPs can induce high antibody titers and elicit neutralizing humoral responses to block EBV infection. More rational and effective designs are required to promote the gp42-N terminal region to become an epitope-based vaccine.