技术资料

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous disorder characterized by impaired mucociliary clearance due to defects in motile cilia. This study investigates the impact of loss-of-function mutations in the CFAP300 gene on the ciliary structure and function in three PCD patients. Using a multimodal approach,we integrated molecular genetic testing,transmission electron microscopy,the high-speed video microscopy assay and immunofluorescence staining to analyze ciliary motility and protein expression in both ex vivo and in vitro-obtained ciliary cells. Our results revealed that the pathogenic variant c.198_200delinsCC (p.Phe67ProfsTer10) in CFAP300 led to the absence of the functional CFAP300 protein,the complete loss of outer and inner dynein arms and immotile cilia. Air–liquid interface (ALI)-cultured cells from patients exhibited no ciliary beating,contrasting with healthy controls. Immunostaining confirmed the absence of CFAP300 in patient-derived cilia,underscoring its critical role in dynein arm assembly. These findings highlight the diagnostic utility of ALI cultures combined with functional and protein analyses for PCD,offering a clinically actionable framework that can be readily incorporated into standard diagnostic workflows. View Publication

Emerging evidence suggests brain-resident myeloid cells,including perivascular macrophages and microglia,provide a reservoir for HIV infection in the central nervous system (CNS),and their inflammatory activation is a proposed pathogenic mechanism in HIV-associated neurocognitive disorders (HAND). We investigated whether cannabinoid receptor 2 (CB2),an immunomodulatory receptor expressed in myeloid cells,regulates viral replication and inflammation in HIV-infected macrophages and microglia. Using the synthetic CB2-specific agonist JWH-133,we found that CB2 activation reduced HIV replication in primary human monocyte-derived macrophages (MDMs) and human induced pluripotent stem cell-derived microglia (iMg) at differing doses,corresponding to the basal expression of CNR2,which encodes CB2,and related endocannabinoid transcripts in each cell type. JWH-133 broadly reduced release of cytokines from HIV-infected MDMs but not iMg. RNA-seq revealed that CB2 agonism primarily altered interferon and integrated stress response pathways in MDMs while altering homeostatic pathways,including synapse maintenance and phagocytosis,in iMg. Further analyses in iMg revealed that NLRP3 inflammasome activation,but not priming,was reduced by CB2 activation,which did not inhibit HIV-induced nuclear factor kB activation. This study identifies key differences in CB2 response between myeloid lineage cell types and implicates CB2-specific agonists as promising candidates for the regulation of HIV-associated neuroinflammation. View Publication

Solid tumor malignancy (STM) patients experience increased risk of breakthrough SARS-CoV-2 infection owing to reduced COVID-19 vaccine immunogenicity. However,the underlying immunological causes of impaired neutralization remain poorly characterized. Furthermore,non-neutralizing antibody functions can contribute to reduced disease severity but remain understudied within high-risk populations. We dissected polyfunctional antibody responses in STM patients and age-matched controls who received adenoviral vector- or mRNA-based COVID-19 vaccine regimens. Elevated inflammatory biomarkers,including agalactosylated IgG,interleukin (IL)-6,IL-18,and an expanded population of CD11c−CD21− double negative 3 (DN3) B cells were observed in STM patients and were associated with impaired neutralization. In contrast,mRNA vaccination induced Fc effector functions that were comparable in patients and controls and were cross-reactive against SARS-CoV-2 variants. These data highlight the resilience of Fc functional antibodies and identify systemic inflammatory biomarkers that may underpin impaired neutralizing antibody responses,suggesting potential avenues for immunomodulation via rational vaccine design. View Publication

Reactivation of the latent viral reservoirs is crucial for a cure of HIV/AIDS. However,current latency reversing agents are inefficient,and the endogenous factors that have the potential to reactivate HIV in vivo remain poorly understood. To identify natural activators of latent HIV-1,we screened a comprehensive peptide/protein library derived from human hemofiltrate,representing the entire blood peptidome,using J-Lat cell lines harboring transcriptionally silent HIV-1 GFP reporter viruses. Fractions potently reactivating HIV-1 from latency contained human Retinol Binding Protein 4 (RBP4),the carrier of retinol (Vitamin A). We found that retinol-bound holo-RBP4 but not retinol-free apo-RBP4 strongly reactivates HIV-1 in a variety of latently infected T cell lines. Functional analyses indicate that this reactivation involves activation of the canonical NF-κB pathway and is strengthened by JAK/STAT5 and JNK signalling but does not require retinoic acid production. High levels of RBP4 were detected in plasma from both healthy individuals and people living with HIV-1. Physiological concentrations of RBP4 induced significant viral reactivation in latently infected cells from individuals on long-term antiretroviral therapy with undetectable viral loads. As a potent natural HIV-1 latency-reversing agent,RBP4 offers a novel approach to activating the latent reservoirs and bringing us closer to a cure. Subject terms: Preclinical research,Infectious diseases View Publication

Alzheimer’s disease (AD) is a devastating neurodegenerative disorder,with no effective treatment currently available. Recently,non-pharmacological therapy,especially gamma frequency stimulation has shown promising therapeutic effects in Alzheimer’s disease (AD) mouse models. Electric field (EF) is a non-invasive biophysical approach for neuronal protection. However,whether EF is beneficial in AD neuropathology remains unknown. In this study,we exposed the P301S tauopathy mouse model to EF at gamma frequency on the head. We demonstrated that EF treatment significantly improved the cognitive impairments in the P301S mice. This was accompanied by reduced tau pathologies,suppressed microglial activation,neuroinflammation and oxidative stress in the tauopathy mouse brain. Moreover,EF treatment induced cell-specific responses in neural cells,with neurons being more susceptible,followed by microglia and oligodendrocytes. EF also had favorable effects on synaptic protein in neurons,inflammatory response and complement signaling in microglia,and myelination in oligodendrocytes. This study provides strong evidence that EF at gamma frequency may have great potential to be a novel therapeutic intervention for P301S by attenuating neuropathology and offering neuroprotection.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13195-025-01859-8. View Publication

Autoantibodies against envelope (Env) protein encoded by human endogenous retrovirus group K (HERV-K) are prevalent in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE),but it remains unclear which proviruses are responsible for this autoantigen. It also remains poorly understood how the transcription of HERV-K loci is regulated in cells that can produce Env.ResultsWe aligned our neutrophil RNA sequencing data to the new telomere-to-telomere reference genome and found uniquely mapping transcripts from HERV-K101,K102,K104,K108,K109,K117 and ERVK5,of which only K102,K108,and K109 encode an intact Env. Expression of K102 and K108 were higher in SLE than in healthy donors or RA (padj < 0.05). Transcripts from these proviruses increased in response to interferon-α in monocytes and neutrophils from RA patients and healthy donors,but not in SLE,presumably because they have chronically elevated type I interferons in vivo. Indeed,HERV-K expression was significantly higher in SLE patients with high type I interferon gene signature. Tumor necrosis factor-α and other cytokines and TLR ligands also induced HERV-K102 and K108 transcripts. Interferon-α also increased detectable Env protein in monocytes,macrophages,and neutrophils from RA patients. Among the genes for epigenetic silencers of HERV-K,only TRIM28 was significantly decreased in SLE patients with high interferons (padj = 0.00024).ConclusionsOur data establish a role for interferons in maintaining increased HERV-K expression in SLE and suggest that interferons or other cytokines can upregulate HERV-K to similar levels in RA. A transient increase may also accompany normal immune responses,suggesting that endogenous retroviruses may have been co-opted for efficient immune responses.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13100-025-00371-y. View Publication

T cell dysfunction is a pivotal driving factor in autoimmune diseases,yet its underlying regulatory mechanisms remain incompletely understood. The role of long non-coding RNAs (lncRNAs) in immune regulation has gradually been recognized,although their functional mechanisms in T cells remain elusive. This study focuses on lncBADR (LncRNA Branched-chain Amino acids Degradation Regulator),elucidating its mechanism by which it regulates branched-chain amino acids (BCAAs) metabolism to influence T cell effector functions. Mice with specific knockout of lncBADR (T celllncBADR−/−) exhibited markedly ameliorated experimental autoimmune encephalomyelitis (EAE) symptoms. Mechanistic investigations revealed that lncBADR inhibits BCAAs degradation by binding to the enzymes Mccc1 and Pcca,leading to the accumulation of BCAAs within T-cells. This,in turn,activates the mTOR-Stat1 signaling pathway,promoting IFN-γ secretion and exacerbating EAE pathology. In contrast,knockout of lncBADR restored BCAAs degradation,significantly reducing IFN-γ secretion in T cells and suppressing their pathogenic functions. Further studies demonstrated that high-BCAAs feeding partially reversed the protective effects of lncBADR knockout,indicating that lncBADR plays a crucial role in autoimmune inflammation by regulating BCAAs metabolism. This study offers new insights into targeting lncBADR or modulating BCAAs metabolism as potential therapeutic strategies for autoimmune diseases. View Publication

Primary human endothelial cells represent an essential tool to model endothelial dysfunction and to screen interventions for its treatment. Here,we developed a protocol for the synchronous isolation of primary human saphenous vein endothelial cells (HSaVEC),human internal thoracic artery endothelial cells (HITAEC),and human microvascular endothelial cells (HMVEC) from SV and ITA utilized as conduits during coronary artery bypass graft surgery and from subcutaneous adipose tissue excised while providing an access to the heart. Treatment by collagenase type IV and magnetic separation with anti-CD31-antibody-coated beads ensured relatively high efficiency of the isolation (≈60% for HSaVEC,≈50% for HITAEC,and ≈20% for HMVEC) and high purity (≥99%) of isolated ECs within ≈2 weeks (HSaVEC),≈2–3 weeks (HITAEC),and ≈3–4 weeks (HMVEC). A colorimetric assay of cell viability and proliferation,as well as real-time bioimpedance monitoring using the xCELLigence instrument,demonstrated high proliferative activity in HSaVEC,HITAEC,and HMVEC,whilst the in vitro tube formation assay indicated their angiogenic potential. The isolation of HSaVEC,HITAEC,and HMVEC from patients undergoing coronary artery bypass graft surgery is a promising option to investigate endothelial heterogeneity,to interrogate endothelial responses to various stresses,and to pinpoint the optimal approaches for restoring endothelial homeostasis,thereby reproducing them within the bedside-to-bench-to-bedside concept. View Publication

BackgroundHigh densities of tertiary lymphoid structures (TLSs) are associated with improved clinical outcomes in various malignancies,including human papillomavirus (HPV)-associated head and neck squamous cell carcinoma (HNSCC). However,the role of TLSs in shaping antitumor immunity in HPV-induced cervical cancer (CESC) remains unclear. Therefore,we analyzed the density,composition,and prognostic impact of TLSs in patients with CESC as well as patients with HNSCC.MethodsMultiplex immunofluorescence,immunohistochemistry,and spatial transcriptomics were used to analyze TLS density and composition in HNSCC and CESC tissue sections with respect to patient prognosis. The spatial approach was supplemented by flow cytometry-based analysis of the polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) phenotype in freshly resected primary tumor tissues.ResultsAlthough both indications were associated with HPV infection,we confirmed a positive correlation between TLS density and improved overall survival only in patients with HNSCC. The TLS composition differed markedly between HNSCC and CESC samples,with a shift toward high regulatory T cell (Treg) and PMN-MDSC abundance in CESC samples. The highest Treg and PMN-MDSC levels were observed in patients with CESC who died of the disease. CESC-infiltrating PMN-MDSCs showed high arginase 1 expression,which correlated with diminished T-cell receptor (TCR)ζ chain expression in CESC-infiltrating T cells. Additionally,the high number of PMN-MDSCs in TLSs was associated with the absence of HPV-specific T cells in CESC.ConclusionsUnlike in HNSCC,the composition of TLSs,rather than their quantity,was associated with the overall survival of patients with CESC. High numbers of Tregs and PMN-MDSCs infiltrating immature TLSs prevail in patients with CESC who succumbed to the disease and seem to affect tumor-specific immune responses. View Publication

Multiplex gene-edited chimeric antigen receptor (CAR) T-cell therapies face significant challenges,including potential oncogenic risks associated with double-strand DNA breaks. Targeted microRNAs (miRNAs) may provide a safer,functional,and tunable alternative for gene silencing without the need for DNA editing. As a proof of concept for multiplex gene silencing,we employed an optimized miRNA backbone and gene architecture to silence T-cell receptor (TCR) and major histocompatibility complex class I (MHC-I) in mesothelin-directed CAR (M5CAR) T cells. The efficacy of this approach was compared to CD3ζ and β2-microglobulin (β2M) CRISPR/Cas9 knockout (KO) cells. miRNA-expressing cassettes were incorporated into M5CAR lentiviral vectors,enabling combined gene silencing and CAR expression. Antitumor activity was evaluated using in vitro assays and in vivo pancreatic ductal adenocarcinoma models. Silenced (S) M5CAR T cells retained antitumor functionality comparable to,and in some cases exceeding,that of KO cells. In vivo,S M5CAR T cells achieved tumor control with higher persistence and superior metastasis prevention. In vitro assays demonstrated enhanced resistance to alloreactive natural killer (NK) cells and peripheral blood mononuclear cells (PBMCs). Titratable multiplex gene silencing via targeted miRNAs offers an alternative to gene editing for CAR T cells,with potential advantages in potency,persistence,metastasis prevention,and immune evasion for allogeneic products. This strategy may overcome tumor-induced immunosuppression while avoiding the risks associated with DNA double-strand breaks. View Publication