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关键词： fibrinolysis   hepatocellular carcinoma   hemopexin   apoptosis  

摘要： Fibrinolysis plays a crucial role in maintaining coagulation homeostasis, but its functions in hepatocellular carcinoma (HCC) remain poorly understood. This study aimed to develop a fibrinolysis-based molecular classification and prognostic signature for HCC and to identify a key regulatory gene. Using non-negative matrix factorization (NMF), we identified distinct fibrinolysis-related HCC subtypes with specific clinical outcomes and tumor microenvironment characteristics. A six-gene prognostic signature comprising ACAT1, GRHPR, HPX, PCK2, IYD, and PON1 was established through weighted gene co-expression network analysis (WGCNA) and LASSO-Cox regression, which effectively stratified patients into different risk groups across multiple cohorts. Hemopexin (HPX) was identified as the top candidate and functionally validated: HPX overexpression suppressed clonogenicity and migration, promoted apoptosis, and inhibited xenograft tumor growth. RNA sequencing analysis suggested associations between HPX and apoptosis as well as TNF-alpha signaling pathways, which were confirmed through flow cytometry apoptosis assays, mitochondrial membrane potential measurements, and TUNEL staining. Western blot and immunohistochemical analyses further demonstrated that HPX upregulates the Bax/Bcl-2 ratio via the TNF-alpha signaling pathway. This study defines novel molecular subtypes of HCC and reveals that HPX exerts anti-tumor effects through TNF-alpha-mediated mitochondrial apoptosis, characterized by an increased Bax/Bcl-2 ratio.

关键词： CRISPR   MHC I   class I   gene editing   genome editing   immunogenicity   immunotoxicity  

摘要： In vivo genome editing with CRISPR-Cas9 systems is generating worldwide attention and enthusiasm for the possible treatment of genetic disorders. However, the consequences of potential immunogenicity of the bacterial Cas9 protein and the AAV capsid have been the subject of considerable debate. Here, we model the antigen presentation in cells after in vivo gene editing by in vitro transduction of a human cell line with an AAV2 vector that delivers the Staphylococcus aureus Cas9 transgene. Through HLA class I enrichment, peptide elution, and highly sensitive LC-MS interrogation, we identified a highly conserved saCas9-derived T cell epitope in the catalytic domain of the enzyme that is restricted to HLA-A*02:01 and induces CD8+ T cell activation and killing. We conclude that AAV delivery of Cas9 results in presentation of a T cell epitope that can activate CD8+ cells and induce killing of the transduced cell, with important ramifications for in vivo genome editing strategies.

摘要： Objective This study investigates the role of senescent epidermal basal cells in cutaneous lupus erythematosus (CLE) pathogenesis using skin samples from patients with CLE and a mouse model of systemic lupus erythematosus (SLE).Methods Cellular senescence profiling used datasets from the NCBI GEO database and Accelerating Medicines Partnership (AMP) Phase 1-Metro. Gene array data from GSE184989 (CLE, n = 68;control, n = 4), single-cell RNA sequencing data from GSE186476 (CLE, n = 7;control, n = 14), and AMP Phase 1-Metro (SLE, n = 17) were used. In vitro experiments further examined the expression of p21WAF1/CIP1, Type I interferon (IFN), HLA class I (HLA-I), and epidermal growth factor receptor (EGFR) signaling. Pharmacological clearance of senescent cells was performed using the senolytic drug fisetin in the SLE mouse *** p21WAF1/CIP1-high senescent epidermal basal cells in patients with CLE exhibit high Type I IFN expression. These cells enhance IFN signaling in surrounding normal epidermal basal cells, leading to increased HLA-I expression. In contrast, senescent epidermal cells up-regulate epidermal growth factor receptor signaling, which downregulates HLA-I expression, allowing them to evade immune surveillance. This heterogeneity of HLA-I expression promotes CD8-positive T-mediated toxicity against normal epidermal basal cells, resulting in their apoptosis. Pharmacological clearance of senescent epidermal basal cells improved SLE-like skin *** Senescent cells create a microenvironment that directs cytotoxic T cell-mediated responses against normal epidermis in patients with CLE, contributing to disease pathology. Targeting senescent cells and their signaling pathways may offer novel therapeutic strategies for patients with skin lesions from CLE and SLE.

关键词： Infectious disease and host defense   Innate immunity and inflammation   Mucosal immunology  

摘要： The ocular surface is a mucosal barrier tissue colonized by commensal microbes, which tune local immunity by eliciting IL-17 from conjunctival gamma delta T cells to prevent pathogenic infection. The commensal Corynebacterium mastitidis (C. mast) elicits protective IL-17 responses from conjunctival V gamma 4 T cells through a combination of gamma delta TCR ligation and IL-1 signaling. Here, we identify V gamma 6 T cells as a major C. mast-responsive subset in the conjunctiva and uncover its unique activation requirements. We demonstrate that V gamma 6 cells require both extrinsic (via dendritic cells) and intrinsic TLR2 stimulation for optimal IL-17A response. Mechanistically, intrinsic TLR2 signaling was associated with epigenetic changes and enhanced expression of genes involved in fatty acid oxidation to support Il17a transcription. We identify a key transcription factor, I kappa B zeta, which is upregulated by TLR2 stimulation and is essential for this program. Our study highlights the importance of intrinsic TLR2 signaling in driving metabolic reprogramming and production of IL-17A in microbiome-specific mucosal gamma delta T cells.

关键词： schizophrenia   treatment-resistant   biomarker   inflammation   apoptosis   interleukin 1 beta   brain-derived neurotrophic factor   Fas ligand   clozapine  

摘要： Background/Objectives: Pro-inflammatory, neurotrophic, and proapoptotic factors affect the course of schizophrenia;however, their impact on the clinical response during relapse is not well recognized. A member of TNF family, Fas ligand (FasL), participates in apoptosis, but its connection with treatment-resistant schizophrenia is unknown. Methods: For this preliminary exploratory study, 53 patients with schizophrenia relapse and 45 healthy subjects were enrolled. Pro-inflammatory interleukin IL-1 beta, brain-derived neurotrophic factor (BDNF), FasL levels, and clinical evaluations (PANSS, SANS, SAPS) were studied at admission, after a 4-week therapy, and at remission. Results: In the clozapine-treated therapy-resistant group, IL-1 beta correlated negatively with clinical improvement (admission, 4-week treatment). In patients not treated with clozapine, IL-1 beta correlated negatively with disease duration (admission). A negative correlation occurred between FasL and clinical improvement in general symptoms (admission, 4-week treatment), FasL and leukocyte count (admission), and IL-1 beta and BDNF levels (4-week treatment). In the clozapine-treated group, the negative correlation between FasL levels and the leukocyte count was absent. Conclusions: The severity of psychopathology in patients with schizophrenia seems to correlate with higher IL-1 beta and lower BDNF. The novelty of our findings is the observation that higher FasL is negatively associated with the degree of clinical improvement. Thus, a decline of FasL during treatment may be proposed as a predictor of clinical recovery. With caution, we suggest that clozapine use may be linked to a protective effect against FasL signaling and the alleviation of apoptotic processes.

关键词： MHC I upregulation   redox-responsive nanovesicles   tyrosine kinase inhibitors   sunitinib   sorafenib  

摘要： Introduction Cancer vaccines work through activating tumor-specific T cells, which can specifically attack cancer cells by recognizing antigens binding with Major-Histocompatibility-Complex I (MHC I) molecules. The downregulation or loss of MHC I expression on tumor cells can affect the efficacy of cancer *** Herein, to increase the MHC I expression on tumor cells, a nanovesicle-based strategy was developed to improve the efficacy of cancer vaccines. Several clinically applied medicines, such as tyrosine kinase inhibitors (TKIs), were screened for their capacity to upregulate MHC *** Two TKIs, Sunitinib and Sorafenib, were found to be very effective in elevating MHC I expression, and they were encapsulated into redox-responsive nanovesicles respectively (SUN-KD10 or SOR-KD10), which demonstrated favourable tumor-targeting capabilities in the tumor microenvironment. Sunitinib or Sorafenib activates the IFN gamma/STAT1 pathway, which improve the expression of MHC I. When combined with whole-tumor-antigen-loaded nanovaccines, these nanovesicle formulations elicited a synergistic antitumor effect in both breast cancer and melanoma mouse models. The tumors in the tumor-bearing mice treated with combined strategy grew more slowly and the survival times of such mice are significantly *** The studies demonstrated that more tumor-specific T cells were activated in the combined strategy treated mice, suggesting improved immune-mediated tumor clearance. This combinatorial approach provides a promising strategy to overcome immune evasion and to enhance the therapeutic outcomes of cancer vaccine-based immunotherapy by using clinical-applied medicines with cancer vaccines.

关键词： Cytokines   COVID 19   Chemokines   SARS CoV 2   Immune response   Enzyme-linked immunoassays   Blood plasma   Respiratory infections  

摘要： Introduction COVID-19 was less severe in Sub-Saharan Africa (SSA) compared with Europe and North America. It is unclear whether these differences could be explained immunologically. Here we determined levels of ex vivo SARS-CoV-2 peptide-specific IFN-gamma producing cells, and plasma cytokines and chemokines over the first month of COVID-19 diagnosis among Kenyan COVID-19 patients from urban and rural areas. Methods Between June 2020 and August 2022, we recruited and longitudinally monitored 188 COVID-19 patients from two regions in Kenya, Nairobi (urban, n = 152) and Kilifi (rural, n = 36), with varying disease severity - severe, mild/moderate, and asymptomatic. IFN-gamma secreting cells were enumerated at 0-, 7-, 14- and 28-days post diagnosis by an ex vivo enzyme-linked immunospot (ELISpot) assay following in vitro stimulation of peripheral blood mononuclear cells (PBMCs) with overlapping peptides from several SARS-CoV-2 proteins. A multiplexed binding assay was used to measure levels of 22 plasma cytokines and chemokines. Results Higher frequencies of IFN-gamma-secreting cells against SARS-CoV-2 spike peptides were observed on the day of diagnosis among asymptomatic compared to patients with severe COVID-19. Higher concentrations of 17 of the 22 cytokines and chemokines measured were positively associated with severe disease, particularly interleukin (IL)-8, IL-18 and IL-1ra (p < 0.0001), while a lower concentration of SDF-1 alpha was associated with severe disease (p < 0.0001). Concentrations of 8 and 16 cytokines and chemokines including IL-18 were higher among Nairobi asymptomatic and mild patients compared to their respective Kilifi counterparts. Conversely, concentrations for SDF-1 alpha were higher in rural Kilifi compared to Nairobi (p = 0.012). Conclusion In Kenya, as seen elsewhere, pro-inflammatory cytokines and chemokines were associated with severe COVID-19, while an early IFN-gamma cellular response to overlapping SARS-CoV-2 spike peptides was assoc

摘要：

关键词： A2CAR   CRISPR/Cas9   EngTreg   GvHD   IL-2-CISC   antigen-specific suppression   dual-HDR editing   engineered Treg   graft versus host disease  

摘要： Chimeric antigen (Ag) receptor-expressing T regulatory cells (CAR-Treg) offer therapeutic potential for treating autoimmunity, allograft rejection, and graft-versus-host disease (GvHD). HLA-A*02 (A2) CAR (A2CAR)-expressing natural Treg have shown efficacy in preclinical models and are being evaluated in phase 1/2 trials. In the current study, we utilized homology-directed-repair (HDR)-based gene editing to generate A2CAR-expressing engineered Treg (EngTreg). HDR at the FOXP3 locus in bulk CD4+ T cells was used to enforce stable co-expression of endogenous FOXP3 and a chemically inducible interleukin (IL)-2 signaling complex (CISC or IL-2 CISC). A2CAR expression was achieved by lentiviral transduction or via dual-HDR editing targeting A2CAR to the TRAC locus. A2CAR+ CISC+ EngTreg (A2CAR EngTreg) products displayed a Treg immunophenotype, low secretion of pro-inflammatory cytokines in response to stimulation, and low cytotoxicity toward A2+ target cells in vitro. In a xenogeneic GvHD model driven by human A2+ peripheral blood mononuclear cells, A2CAR EngTreg showed superior therapeutic efficacy compared with polyclonal EngTreg. Further, in vivo activation of the IL-2 CISC improved efficacy at limiting doses of A2CAR EngTreg. Together, these findings demonstrate efficient generation of Ag-specific EngTreg utilizing CAR as the targeting moiety and efficacy of A2CAR EngTreg in preclinical models, suggesting potential therapeutic benefit for CAR-expressing EngTreg in transplantation and autoimmune diseases.

关键词： 骨髓来源的巨噬细胞(BMDM)   锌指蛋白Krüppel样转录因子9(KLF9)   1型单纯疱疹病毒(HSV-1)   机制  

摘要： 目的 明确锌指蛋白Krüppel样转录因子9（KLF9）在1型单纯疱疹病毒（HSV-1）诱导巨噬细胞I型干扰素表达中的作用及机制。方法 通过琼脂糖凝胶电泳、实时荧光定量PCR及Western blot法检测KLF9在Klf9-/-（gKO）小鼠骨髓来源的巨噬细胞（BMDM）中的敲除效果；RNA-seq 筛选gKO小鼠及对照小鼠Klf9+/+（WT）BMDM中HSV-1刺激后Klf9下游关键差异基因；实时荧光定量PCR验证HSV-1刺激后干扰素β1（Ifnb1）及下游干扰素刺激基因（ISG）如干扰素诱导蛋白与四肽重复 1（Ifit1）、干扰素刺激的核酸外切酶基因 20（Isg20）、胆固醇25-羟化酶（Ch25h）及2'-5'-寡聚腺苷酸合成酶（Oasl1）的表达情况；ELISA检测HSV-1刺激后gKO及WT小鼠BMDM上清中β干扰素（IFN-β）的含量；Western blot法检测HSV-1刺激后gKO及WT小鼠BMDM中磷酸化干扰素调节因子 3（p-IRF3）、干扰素调节因子 3（IRF3），p-IRF7、IRF7，磷酸化的核因子κB p65（p-NF-κB p65）、核因子κB p65（NF-κB p65）的表达；CUT-Tag及ChIP-qPCR技术检测KLF9在Ifnb1上的结合。结果 KLF9在gKO小鼠的BMDM中表达明显减少；RNA-seq 结果显示BMDM中Klf9缺失导致I型干扰素信号通路受损；实时荧光定量PCR表明Klf9敲除后BMDM中除了Isg20以外，Ifit1、Ch25h及Oasl1表达均明显降低；ELISA显示Klf9敲除导致BMDM分泌的IFN-β明显减少。在机制研究方面，Western blot显示Klf9的缺失不影响p-IRF3、p-IRF7及p-NF-κB p65的表达；CUT-Tag及ChIP-qPCR显示KLF9能直接结合至Ifnb1的启动子区促进其转录。结论 KLF9对巨噬细胞抵抗HSV-1感染至关重要。