Search Results (159)

Background/Objectives: CD19-directed chimeric antigen receptor T (CAR-T) cell therapy induces profound immune remodeling. Nonetheless, biomarkers predicting complete remission (CR) remain poorly defined. We characterized longitudinal cytokine and immune-cell dynamics after CAR-T infusion and identified early immunological features associated with CR. Methods: Longitudinal immune profiling was performed in 18 patients with non-Hodgkin lymphoma, including 14 with relapsed/refractory diffuse large B-cell lymphoma treated with anti-CD19 CAR-T cells. Peripheral blood was collected at the baseline and days 7, 14, 21, 28, and 60 post-infusion. Multiparameter flow cytometry quantified lymphoid and myeloid subsets and Toll-like receptor (TLR)2 and TLR4 expression. Serum cytokines were measured by multiplex assays. Machine-learning-based feature selection identified variables associated with CR. Results: Two inflammatory waves were observed. The first, at day 7, featured elevated IL-6, IL-10, IFN-α, IFN-γ, and TNF-α, accompanied by increased CD4⁺ T cells, HLA-DR^high classical monocytes, and non-classical monocytes. The second, at days 21–28, showed increased IL-5, IL-6, IL-12, IFN-γ, and GM-CSF, with expansion of CD4⁺ and CD8⁺ T cells, regulatory T cells, NK-T cells, and non-classical monocytes. TLR2 expression was significantly upregulated at day 7 on T-cell subsets and on classical and intermediate monocytes. An exploratory feature-selection analysis identified baseline and day-7 TLR2 and TLR4 expression on lymphoid and myeloid cells, early IFN-γ levels, and monocyte frequencies as variables associated with CR. Conclusions: Together, these data show that anti-CD19 CAR-T therapy induces two coordinated waves of cytokine release and immune-cell activation. Moreover, the findings suggest that early modulation of innate immune features, particularly TLR2 expression, is associated with complete remission, although these biomarker relationships remain exploratory and require validation in larger cohorts. Full article

Toll-like receptor 7 (TLR7) agonism offers a promising avenue for antiviral intervention. This study characterizes AXC-715, a novel small-molecule agonist that selectively targets TLR7 to elicit broad-spectrum antiviral effects. Structural analysis of the AXC-715–hTLR7 complex (PDB ID: 5GMH) elucidates the molecular basis of receptor activation. AXC-715 occupies the interface of TLR7 monomers, establishing critical hydrogen bonds with D555 and T586, alongside π-π and π-alkyl interactions with F408, V381, and L557. These interactions effectively promote and stabilize the active TLR7 dimeric conformation. Functionally, AXC-715 activates NF-κB signaling in a P65-dependent manner without inducing cytotoxicity in PK-15 or THP-1 cells. In vitro assays demonstrated that AXC-715 potently inhibits the replication of both pseudorabies virus (PRV) and vesicular stomatitis virus (VSV) by specifically impairing viral replication, distinct from adsorption, entry, assembly, or release processes. The antiviral effect was abolished in TLR7-knockout PK-15 cells, confirming the strict dependence of AXC-715 on on-target TLR7 signaling. These findings highlight AXC-715 as a potent TLR7 agonist that stabilizes receptor dimerization to inhibit viral replication, providing a valuable framework for developing TLR7-based antiviral therapeutics. Full article

Background: Hepatic ischemia–reperfusion (I/R) injury induces systemic oxidative stress and inflammatory responses that may lead to remote lung injury. This study investigated whether taxifolin attenuates hepatic I/R-induced lung damage and examined the involvement of the nuclear factor-κB (NF-κB) and high-mobility group box-1 (HMGB1) signaling axis. Methods: Twenty-eight male Wistar rats were divided into four groups (n = 7): control, taxifolin, hepatic I/R, and taxifolin+I/R. Serum oxidative stress markers (malondialdehyde [MDA], interleukin [IL]-6, total antioxidant/oxidant status [TAS/TOS]) and wet-to-dry lung weight ratio were measured. Lung tissues were evaluated histopathologically and immunohistochemically for NF-κB and HMGB1 expression. Bioinformatics pathway enrichment and molecular docking analyses were also performed. Results: Hepatic I/R significantly increased serum MDA, IL-6, and TOS levels and decreased TAS (p < 0.05). Severe lung injury was observed in the hepatic I/R group (median score: 11), whereas taxifolin pretreatment significantly reduced the injury score (median score: 5, p < 0.001). NF-κB and HMGB1 expression were markedly elevated following hepatic I/R and significantly decreased with taxifolin treatment (p < 0.05). A strong positive correlation was found between NF-κB and HMGB1 expression (r = 0.82, p < 0.001). Pathway enrichment analysis indicated involvement of Toll-like receptor (TLR)-related inflammatory signaling, and docking analysis demonstrated favorable binding of taxifolin to TLR4 and NF-κB p65. Conclusion: Taxifolin attenuated hepatic I/R-induced lung injury by reducing oxidative stress and suppressing HMGB1–TLR4–NF-κB-mediated inflammatory signaling. Full article

Mycoplasma pneumoniae (M. pneumoniae) is the crucial factor of global acquired respiratory infections. Currently, there are no specific disease modification treatments or vaccines available, and the vaccine development for this pathogen lags behind due to the complexity and variability of its antigens. A novel vaccine with broad-spectrum characteristics is essential to provide comprehensive protection against continuously evolving wild-type strains. Here, a broad-spectrum muti-epitope vaccine against M. pneumoniae had been designed through immunoinformatics methods. To ensure its broad-spectrum, we generated consistent sequences from all the antigen proteins of different strains, and then identified potential T cell epitopes. The multi-epitope vaccine (MEV) of M. pneumoniae incorporated 16 CTLs and 7 HTLs from the HMW1–3 and p1 adhesin proteins, which comprised 458 amino acids with adjuvant. The vaccine evaluation showed that the MEV had ideal physicochemical properties, high antigenicity, high immunogenicity, and was non-toxic. Furthermore, there was a strong and stable binding interaction between this vaccine and the toll-like receptors, which could be supported by the normal mode analysis. Finally, codon optimization resulted in the optimal GC content and higher CAI value. The vaccine candidate is expected to induce strong cellular immune responses and may provide protective immunity against the pathogen. We provided a novel in silico vaccine design strategy for vaccine design, which could provide a technical framework for the development of vaccines against other pathogens. Full article

Background/Objectives: Brachytherapy (BT) is a local radiation treatment method for solid tumors. A single 10 Gy high–dose–rate (HDR) BT acts as an “in situ” vaccination. Tumor microenvironment (TME)–dependent radio–resistance mechanisms, such as increasing immunosuppression and hypoxia, lead to tumor recurrence after radiotherapy. Our study aimed to determine whether adding imiquimod (IMQ) to anticancer therapy would overcome TME–mediated mechanisms of radiotherapy resistance. IMQ, a toll–like receptor 7 (TLR7) agonist, acts as an immunostimulant and a vascular normalizing agent. Methods: Mice with well–developed tumors were treated with IMQ at a vascular–normalized dose of 50 μg, followed 5 days later by a single 10 Gy HDR BT. The dose coverage was planned using Discovery RT computed tomography CT scans. Irradiation was performed with a high–dose–rate afterloader equipped with an iridium–192 radioactive source. Results: In mice treated with a combination of IMQ and BT, we observed significant inhibition of melanoma tumor growth. We also noticed an effective therapeutic effect in mice with breast cancer, resulting in significantly prolonged survival and complete tumor regression in 20% of treated mice. In the blood of treated mice, we observed leukopenia with eosinophilia. In tumors, there was enhanced infiltration by cytotoxic CD8⁺ T lymphocytes. The depletion of CD8⁺ T cells completely abolished the effect of the combined therapy. Conclusions: The combination of IMQ with HDR brachytherapy induces a synergistic effect, improving the therapeutic antitumor effect of brachytherapy. Our data indicate that it is reasonable to use drugs that prevent changes in the TME in combination with radiotherapy. Full article

This study investigates the genomic basis of immune adaptation in the transparent glass catfish (Kv: Kryptopterus vitreolus), focusing on the loss of the Toll-like receptor 21 (TLR21) gene. Comparative genomic analysis with closely related non-transparent North African catfish (Cg: Clarias gariepinus) revealed 11 TLR genes in the latter, while only 8 TLR genes (KvTLR1, 2, 3, 5, 7, 9, 13, and 20) were retained in the glass catfish, with TLR21 specifically absent. Collinearity analysis confirmed that the genomic region containing TLR21 is conserved across eight siluriform species, with loss exclusively in the glass catfish, supporting its lineage-specific absence. Structural expansion was notable in KvTLR5, KvTLR7, and KvTLR20. Molecular docking indicated that binding stability between CpG oligonucleotides and TLR21 varies significantly, with CpG-B 1681 showing the strongest interaction, which highlights sequence-dependent ligand recognition. Interestingly, absence of the TLR1 gene in another transparent teleost, the X-ray tetra (Pristella maxillaris), suggests that transparent fishes may share an evolutionary trend of lineage-specific TLR gene loss. Together, these findings reveal a distinctive evolutionary trajectory in the innate immune receptor family of transparent fishes and provide new molecular insights into their adaptive immune strategies. These insights will benefit the academic community by improving comparative frameworks for fish innate immunity, and they may inform disease prevention and health management strategies in aquaculture and the ornamental fish trade. Full article

Sharp declines in temperature pose a significant risk for mass mortality events in the Chinese soft-shelled turtle (Pelodiscus sinensis). To assess the effects of acute cold stress on intestinal health, turtles were exposed to temperatures of 28 °C (control), 14 °C, and 7 °C for 1, 2, 4, 8, and 16 days. The results showed that acute cold stress at 14 °C and 7 °C induced time-dependent alterations in intestinal morphology and histopathology. The damage was more severe at 7 °C, characterized by inflammatory cell infiltration, lymphoid hyperplasia, and extensive detachment and necrosis across the villi, muscle layer, and submucosa. 16S rDNA sequencing revealed significant shifts in intestinal microbiota composition in the 7 °C group, dominated by Helicobacter and Citrobacter. Transcriptomic analysis identified differentially expressed genes (DEGs) that respond to acute cold stress and are involved in the Toll-like receptor signaling pathway (Tlr2, Tlr4, Tlr5, Tlr7, and Tlr8), the NOD-like receptor signaling pathway (Traf6, Traf2, Casr, Rnasel, Pstpip1, Plcb2, Atg5, and Mfn2), apoptosis (Tuba1c, Ctsz, Ctsb, Kras, Hras, Pik3ca, Bcl2l11, Gadd45a, Pmaip1, Ddit3, and Fos), and the p53 signaling pathway (Serpine1, Sesn2, Ccng2, Igf1, Mdm2, Gadd45a, Pmaip1, and Cdkn1a). Metabolomic profiling highlighted differentially expressed metabolites (DEMs) that cope with acute cold stress, such as organic acids (oxoglutaric acid, L-aspartic acid, fumaric acid, DL-malic acid, and citric acid) and amino acids (including L-lysine, L-homoserine, and allysine). The integrated analysis of DEGs and DEMs underscored three key pathways modulated by acute cold stress: linoleic acid metabolism, neuroactive ligand–receptor interaction, and the FoxO signaling pathway. This study provides a comprehensive evaluation of intestinal health in Chinese soft-shelled turtles under acute cold stress and elucidates the underlying mechanisms. Full article

Background and Objectives: Toll-like receptors (TLRs) are pattern recognition receptors with an essential role in regulating both the innate and adaptive immune response. Given their pleiotropic effects in mounting an immune response, previous studies have proposed targeting these TLRs might render alternative strategies for cancer therapy. Synthetic immune response modifiers, such as imidazoquinolines, stimulate the immune cells by activating Toll-like receptors, particularly TLR7/8 receptors, consequently mounting an immune response. Agonists of this class activate, via TLR-mediated signaling, dendritic and B cells, as well as myeloid cells and T cells, thus exhibiting good prospects for cancer immunotherapy. In the present study, we sought to evaluate the effect of imiquimod and gardiquimod, two TLR 7 and 7/8 agonists, respectively, on tumor growth and phenotype of NK cells associated with melanoma. Materials and Methods: We generated a syngeneic model of melanoma in C57BL/6J mice by subcutaneously injecting murine melanoma cells and monitoring tumor growth. Starting on day 8 or 14, we applied TLR agonists either intratumorally or topically and followed the tumor dynamics and NK cell-associated pattern. Results: Our results suggest that both TLR agonists displayed an antitumor effect along with a phenotypically activated profile of NK cells. Both imiquimod and gardiquimod treatment inhibited tumor growth, with gardiquimod showing an increased potency compared to imiquimod. Conclusions: This implies that TLR agonists like imiquimod and gardiquimod could serve as neoadjuvant, adjuvant, or complementary immunotherapeutic agents in melanoma therapy. Full article

Toll-like receptor 7 (TLR7) and Stimulator of Interferon Genes (STING) ligands possess a series of immunomodulatory effects such as anti-infection, anti-tumor, and autoimmune-disease-alleviating effects. In this study, porcine TLR7 (pTLR7) and porcine STING (pSTING) were selected as targets, and molecular docking and virtual screening methods were used for screening of dual-target livestock immunomodulators. Finally, two compounds were screened with molecular docking scores higher than the positive control compounds. They have good binding ability with pTLR7 and pSTING proteins, as well as satisfactory predictive safety and pharmacokinetic properties. Molecular dynamics (MD) simulation results also indicated that the above ligands can form stable complexes with two target proteins. The average binding free energies of compound 2 with pTLR7 and pSTING were −28.65 kcal/mol and −30.12 kcal/mol, respectively, and of compound 7 with pTLR7 and pSTING were −35.93 kcal/mol and −31.70 kcal/mol, respectively, which were comparable to that of positive control ligands. The similarity of target proteins between pigs, humans, and mice, as well as the interactions between ligands and TLR7 and STING in different species, were analyzed. And analysis of predicted structure–activity relationship (SAR) was conducted. Briefly, compound 2 and compound 7 were predicted to form stable complexes with pTLR7 and pSTING, with satisfactory predicted physicochemical properties and pharmacokinetic characteristics, and represented candidates for experimental validation. This study supplies a research basis for the development, design, and structural modification of immune enhancers for animals. Full article

Astrocytes and microglia constitute nearly half of all central nervous system cells and are indispensable for its proper function. Both exhibit striking morphological and functional heterogeneity, adopting either neuroprotective (A2, M2) or proinflammatory (A1, M1) phenotypes in response to cytokines, pathogen-associated molecular patterns (PAMPs)/damage-associated molecular patterns (DAMPs), toll-like receptor 4 (TLR4) activation, and NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome signaling. Crucially, many of these phenotypic transitions arise during the earliest stages of neurodegeneration, when glial dysfunction precedes overt neuronal loss and may act as a primary driver of disease onset. This review critically examines glial-centered hypotheses of neurodegeneration, with emphasis on their roles in early disease phases: (i) microglial polarization from an M2 neuroprotective state to an M1 proinflammatory state; (ii) NLRP3 inflammasome assembly via P2X purinergic receptor 7 (P2X7R)-mediated K⁺ efflux; (iii) a self-amplifying astrocyte–microglia–neuron inflammatory feedback loop; (iv) impaired microglial phagocytosis and extracellular-vesicle–mediated propagation of β-amyloid (Aβ) and tau; (v) astrocytic scar formation driven by aquaporin-4 (AQP4), matrix metalloproteinase-9 (MMP-9), glial fibrillary acidic protein (GFAP)/vimentin, connexins, and janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) signaling; (vi) cellular reprogramming of astrocytes and NG2 glia into functional neurons; and (vii) mitochondrial dysfunction in glia, including Dynamin-related protein 1/Mitochondrial fission protein 1 (Drp1/Fis1) fission imbalance and dysregulation of the sirtuin 1/peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Sirt1/PGC-1α) axis. Promising therapeutic strategies target pattern-recognition receptors (TLR4, NLRP3/caspase-1), cytokine modulators (interleukin-4 (IL-4), interleukin-10 (IL-10)), signaling cascades (JAK2–STAT, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), phosphoinositide 3-kinase–protein kinase B (PI3K–AKT), adenosine monophosphate-activated protein kinase (AMPK)), microglial receptors (triggering receptor expressed on myeloid cells 2 (TREM2)/spleen tyrosine kinase (SYK)/ DNAX-activating protein 10 (DAP10), siglec-3 (CD33), chemokine C-X3-C motif ligand 1/ CX3C motif chemokine receptor 1 (CX3CL1/CX3CR1), Cluster of Differentiation 200/ Cluster of Differentiation 200 receptor 1 (CD200/CD200R), P2X7R), and mitochondrial biogenesis pathways, with a focus on normalizing glial phenotypes rather than simply suppressing pathology. Interventions that restore neuroglial homeostasis at the earliest stages of disease may hold the greatest potential to delay or prevent progression. Given the complexity of glial phenotypes and molecular isoform diversity, a comprehensive, multitargeted approach is essential for mitigating Alzheimer’s disease and related neurodegenerative disorders. This review not only synthesizes pathogenesis but also highlights therapeutic opportunities, offering what we believe to be the first concise overview of the principal hypotheses implicating glial cells in neurodegeneration. Rather than focusing on isolated mechanisms, our goal is a holistic perspective—integrating diverse glial processes to enable comparison across interconnected pathological conditions. Full article

Advanced glycation end products (AGEs) are compounds that accumulate in hyperglycemic states, contributing significantly to the development of diabetes and its complications, including the exacerbation of periodontal disease. We hypothesized that AGEs affect the expression of inflammatory mediators in gingival cells, thus contributing to the increased severity of periodontitis observed in diabetic patients. Thus, we stimulated the gingival epithelial carcinoma-derived cell line, Ca9-22, with AGEs and examined their effect on the expression of prostaglandin E₂ (PGE₂) and its primary synthesizing enzyme, cyclooxygenase 2 (COX2), key inflammatory mediators in periodontitis. AGEs significantly increased the expression levels of COX2 (n = 6, p < 0.001) and the production of PGE₂ (n = 5, p < 0.05) compared to untreated control and bovine serum albumin (BSA) groups. The receptor for AGEs (RAGE) inhibitor FPS-ZM1 blocked the AGEs-stimulatory effects on COX2 (n = 7, p < 0.01), PGE₂ (n = 6, p < 0.001), and Toll-like receptor 4 (TLR4) expression (n = 7, p < 0.001). Furthermore, AGEs induced the phosphorylation of protein kinase C (p-PKC) via the TLR4 pathway (n = 7, p < 0.01). Crucially, AGEs enhanced NF-κB nuclear accumulation, which was inhibited by blocking either RAGE (n = 5, p < 0.0001) or TLR4 (n = 5, p < 0.0001). In conclusion, these findings demonstrate that AGEs increase PGE₂ production in Ca9-22 cells primarily through a signaling cascade involving RAGE and the TLR4-PKC-NF-κB pathway. Our results suggest TLR4 as a critical mediator that contributes to AGEs-induced inflammation. Full article

Sepsis is a clinical syndrome caused by abnormal host response to infection. Thrombocytopenia and platelet dysfunction are common findings in sepsis and associated with worse outcomes. The innate immune single-stranded RNA sensor, Toll-like Receptor-7 (TLR7), plays a key role in thrombocytopenia in sepsis. This study investigated whether TLR7 signaling also contributes to platelet dysfunction in sepsis, and whether the bioactivity of downstream inflammatory mediators, specifically extracellular vesicles (EVs), is impacted by the TLR7 signaling pathway. Sepsis was induced in wild-type (WT) and TLR7-deficient (TLR7^−/−) mice by cecal ligation and puncture. Blood was collected at twenty-four hours for platelet and plasma isolation, and platelet function was assessed using aggregation, adhesion, and calcium flux assays. EVs were isolated from plasma and used in vitro to evaluate their impact on platelet–leukocyte aggregate (PLA) formation. We found that septic platelets are highly activated and more adhesive, yet show markedly impaired aggregation and reduced calcium signaling, indicating functional exhaustion despite activation. Notably, mice lacking TLR7 maintained stronger platelet aggregation, enhanced adhesion, and preserved calcium release in the septic state compared to wild-type controls, suggesting a protective effect of TLR7 deficiency. Plasma EVs increased in abundance and size during sepsis and promoted clot and PLA formation in vitro. Notably, EV-mediated platelet activation was reduced with EVs derived from TLR7-deficient mice. Our results demonstrate that while sepsis drives persistent platelet activation and dysfunction, TLR7 deficiency preserves platelet function and modulates the pathogenic activity of EV-mediated platelet activation, highlighting TLR7 as a key regulator and potential therapeutic target in sepsis-induced platelet dysfunction. Full article

Plasmacytoid dendritic cells (pDCs) are a unique subset of dendritic cells specialized in rapid and robust type I interferon (IFN) production, playing critical roles in the pathogenesis and pathomechanisms of many human diseases. Accurate identification of pDCs in peripheral blood mononuclear cells (PBMCs) is challenging due to dynamic and non-exclusive specific expression of surface markers such as blood dendritic cell antigen (BDCA)-2 and BDCA-4. Although BDCA-4 is generally more stably expressed than BDCA-2, prolonged stimulation or inflammatory conditions can induce its expression on multiple non-pDC cell types, reducing the accuracy of pDC identification. Here, we thoroughly investigated BDCA-4 expression dynamics on pDCs and other PBMC subsets following prolonged activation with Toll-like receptor (TLR) 7 and TLR9 agonists. Our flow cytometry analysis revealed a significant increase in BDCA-4-positive non-pDC populations after extended stimulation, primarily corresponding to CD14⁺ monocytes. To overcome this limitation, we performed a gating strategy combining BDCA-4 positivity with a cocktail of non-pDC markers, enabling the exclusion of non-pDCs and accurate identification of pDCs. This approach enables the reliable identification of pDCs within heterogeneous cell populations using only two fluorescent channels in healthy conditions and even during strong activation or pathological states characterized by chronic inflammation. Full article

Targeting Toll-like receptors 7 and 8 (TLR7/8) has emerged as a promising strategy in cancer immunotherapy. TLR7/8 agonists activate robust Th1-type immune responses and bridge innate and adaptive immunity. Further, TLR7/8 agonists can serve as valuable adjuncts to conventional therapies, such as chemotherapy and radiotherapy, enhancing efficacy while reducing adverse effects. Their integration into combination regimens for cancer offers a dual advantage: amplifying antitumor immunity and reducing tumor burden. Notably, the incorporation of TLR7/8 agonists into cancer vaccine platforms has yielded encouraging results in preclinical models and is advancing toward clinical application. This review highlights the mechanisms of action, therapeutic potential, and recent progress in the development of TLR7/8 agonist-based strategies for cancer treatment. We also discuss ongoing clinical evaluations and the rationale for combining these agents with existing modalities to enable more effective, personalized, and accessible cancer therapies. Full article

Multiple sclerosis (MS) is a chronic autoimmune disease characterized by sustained neuroinflammation and demyelination within the central nervous system (CNS). Vesatolimod (VES), a selective Toll-like receptor 7 (TLR7) agonist, has demonstrated both antiviral and immunomodulatory properties; however, its potential therapeutic value in neuroinflammatory contexts remains poorly understood. In this study, we evaluated the efficacy of VES in the experimental autoimmune encephalomyelitis (EAE) model of MS and elucidated its mechanisms of action. EAE was induced in mice by immunization with myelin oligodendrocyte glycoprotein (MOG_35–55). The therapeutic effects of VES were assessed through clinical scoring, body weight monitoring, histopathology, flow cytometry, quantitative proteomics, and Western blot analysis. Additionally, an in vitro model of lipopolysaccharide (LPS)-induced microglial activation was employed to investigate cell-autonomous mechanisms. Results showed that VES administration significantly ameliorated disease severity, reduced weight loss, and enhanced neurological function in EAE mice. Treatment with VES inhibited the differentiation of pro-inflammatory Th1 and Th17 cells while expanding regulatory T cell (Treg) populations. It also preserved blood–brain barrier (BBB) integrity, attenuated demyelination, and modulated microglial activation phenotypes within the CNS. At the molecular level, VES activated the Nrf2/HO-1 antioxidant pathway, thereby enhancing the expression of cytoprotective proteins. Proteomic profiling further revealed the downregulation of inflammation-related proteins, specifically those associated with TNF, IL-17, and NOD-like receptor signaling pathways. Collectively, these findings demonstrate that VES alleviates neuroinflammation in EAE through multimodal mechanisms—including peripheral and central immune regulation, BBB protection, and activation of endogenous antioxidant defenses—supporting its further development as a promising therapeutic candidate for MS. Full article