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While radiotherapy (RT) is effective for local tumor control, it rarely induces the regression of non-irradiated metastases, a phenomenon known as the abscopal effect. The mechanisms constraining this systemic immune response remain poorly understood. This study investigates galectin-9 (Gal-9), a ligand for the TIM-3 (T-cell immunoglobulin and mucin-domain containing-3) immune checkpoint, as a mediator of immune escape that limits RT systemic efficacy and evaluates combinatorial RT/Gal-9 blockade in preclinical models. METHODS: Multiplatform analysis (RNA sequencing, immunoblot, flow cytometry, ELISA, immunohistochemistry) characterized RT-induced Gal-9 regulation in human lung/colorectal cancer cell lines, murine tumors/serums, and paired patient tumors. Local and abscopal therapeutic efficacy was evaluated in homologous (CT26/CT26, LLC/LLC) and heterologous (CT26/4T1) two-tumor mouse models. Immune profiling of tumor microenvironment, tumor-draining lymph nodes (tdLNs), and splenic compartments was comprehensively assessed by flow cytometry. Mechanistic studies employed STING (Stimulator of Interferon Genes) inhibition (H151), CD8+ T-cell depletion (anti-CD8α), macrophage/monocyte depletion (PLX-3397), interferon-I (IFN-I) blockade (anti-IFNAR1), and lymphocyte egress inhibition (FTY720). RT upregulated Gal-9 predominantly within host myeloid compartments (dendritic cells, macrophages, monocytes, neutrophils) versus tumor cells, in both irradiated tumors and abscopal tumors. Mechanistically, RT-activated STING-IFN-I axis locally induced Gal-9+ myeloid cells that subsequently disseminated systemically. Clinically, elevated post-RT Gal-9 in patient biopsies correlated with poor therapeutic outcomes. Notably, combining Gal-9 blockade with RT elicited potent abscopal responses in homologous two-tumor mouse models. Furthermore, anti-Gal-9 markedly enhanced radio-immunotherapy efficacy in the poorly immunogenic Lewis lung carcinoma. Immunologically, Gal-9 blockade synergized with RT to activate the myeloid and T cell compartments, enhancing dendritic cell accumulation in tdLNs and boosting CD8+ T cell infiltration in abscopal tumors. Depletion of CD8+ T cells/monocytes or blocking lymphocyte egress from lymph nodes abrogated the abscopal efficacy, underscoring their essential roles. Our findings establish RT-induced Gal-9 as a novel dual myeloid/T-cell immune checkpoint restricting abscopal responses. Gal-9 blockade represents a promising strategy to potentiate radiotherapy against metastatic disease, defining a therapeutic paradigm distinct from conventional checkpoint inhibitors.

The primary cause of alopecia areata (AA) is autoimmune-mediated hair follicle destruction, while breakdown of immune tolerance due to Treg cell homeostasis disruption critically contributes to this process. However, in AA, the factors leading to Treg cell impairment and the effective regulatory pathways remain unanswered. To assess the therapeutic effects of sCD83 on AA and elucidate the crucial role of sCD83-mediated Treg cell activation in remodeling the perifollicular microenvironment. Blood and scalp tissue were collected from AA patients and healthy controls to characterize sCD83 using ELISA, flow cytometry, and immunofluorescence. In graft-induced C3H/HeJ mouse model of AA, the therapeutic effect of sCD83 on early-onset AA was evaluated by H&E staining, immunofluorescence, and flow cytometry. In vitro, human hair follicle organ culture and primary outer root sheath keratinocyte (ORSK) culture were utilized to clarify the impact of sCD83 on the expression and activity of indoleamine 2,3-dioxygenase (IDO) in hair follicle. Co-culture of ORSKs and PBMCs, together with administration of an IDO inhibitor to AA mice, were performed to determine the necessity of IDO for Treg cell activation. Finally, GST-pulldown and co-IP assays were employed to identify potential sCD83 receptors on ORSKs. We showed here that AA patients exhibit sCD83 deficiency, which may be attributed to reduced sCD83 release from DCs, and that serum sCD83 levels are negatively correlated with disease severity. Supplementation with sCD83 in AA mouse reversed disease manifestations and promoted Treg cell proliferation. Mechanistically, IDO activity in ORSKs is essential for sCD83-mediated Treg cell activation, with TRX potentially serving as a sCD83 receptor to regulate IDO expression. Our study establish the functional role of sCD83 in AA, confirms the therapeutic potential of sCD83 supplementation, and provides some mechanistic insights.

Synovial sarcoma (SS) is a rare and highly malignant soft tissue sarcoma. The efficacy of existing therapies is limited, and new strategies are urgently needed. Osteopontin (OPN) promotes disease progression by regulating tumor-immune cell interaction and immunosuppressive microenvironment, which is a potential therapeutic target. The aim of this study was to investigate the role of OPN in SS immune regulation and tumor progression. A stable cell line with OPN knockdown was constructed using lentiviral transduction. RNA-seq and bioinformatics analysis were used to predict the correlation between OPN and immunity. Flow cytometry and ELISA were used to detect the changes in the number and function of CD8 + T cells. EDU, CCK-8 and Transwell chamber experiments were used to detect the proliferation, invasion and migration ability of SS. The protein interaction between OPN and PD-L1 was detected by molecular docking and Co-IP. The expression of PD-L1 was detected by Western blot and flow cytometry. The expression levels of OPN, PD-L1 and CD8 were detected by immunohistochemistry. To predict the correlation of OPN, PD-L1 and CD8 in sarcoma tissues and their clinical prognostic significance. RNA-seq analysis revealed that OPN-associated differentially expressed genes were significantly enriched in immune response-related biological processes. Bioinformatics analysis demonstrated that OPN expression in sarcoma was associated with immune responses and T-cell activation, and was negatively correlated with immune cell infiltration. OPN induced CD8⁺ T-cell exhaustion, thereby promoting immune evasion and facilitating the proliferation, invasion, and migration of synovial sarcoma (SS) cells. Molecular docking and co-immunoprecipitation assays confirmed a direct protein-protein interaction between OPN and PD-L1, and knockdown of OPN resulted in a significant reduction in PD-L1 expression. In SS tissues, OPN was highly expressed, whereas PD-L1 and CD8 were expressed at low levels and exhibited a positive correlation. Furthermore, bioinformatics analysis showed that PD-L1 expression was positively correlated with CD8 expression in sarcoma, and low CD8 expression was associated with poor prognosis. We preliminarily propose that OPN promotes immune evasion in synovial sarcoma (SS) by regulating PD-L1 expression and subsequently inducing CD8⁺ T-cell exhaustion. This mechanism suggests that OPN may serve as a novel immunotherapeutic target and provides new insights into the development of immunotherapy strategies for SS.

Pancreatic ductal adenocarcinoma (PDAC) growth and metastasis are influenced by the tumor microenvironment (TME), which includes immune cells, endothelial cells, macrophages, and cancer-associated fibroblasts (CAFs). Since the novel integrin-targeted cytotoxin ProAgio can inhibit activated CAFs and endothelial cells, we investigated its combination with standard of care chemotherapies in genetically engineered mouse (GEM) and orthotopic murine models of PDAC. We established metastatic murine KPC-Ganji & Bassel-Luc (mKPC-GB-Luc) cell lines and validated them using bulk RNA sequencing. Two in vivo orthotopic mouse model were used to evaluate ProAgio with chemotherapy. mKPC-GB-Luc was used to evaluate the 5FU, oxaliplatin, and irinotecan (FOI combination), and KPC-ML1-Luc was used to evaluate the gemcitabine, nab-paclitaxel (GPTx combination). Immunohistochemistry was used to measure integrin β3, E-cadherin, and HIF-1α. Hypoxia was evaluated using pimonidazole. Stem cells, CAFs, and immune cell subtypes were quantified by flow cytometry. We evaluated the combination of ProAgio plus gemcitabine in a KPC genetically engineered mouse model (KPC GEM). Two sets of KPC GEM were developed. The first set was used for a survival study. The second set was terminated early and tumors were used for single-cell RNA seq and sequential multiplex immunofluorescence (COMET). Compared with the chemo regimen (GPTx or FOI), ProAgio, or sham, the combination of ProAgio plus chemo significantly modulated the TME, reduced tumor weight, reduced hypoxia, and eliminated metastasis to the lungs and liver. The combination of ProAgio and gemcitabine increased overall survival in KPC GEM mice compared with either treatment alone. Single-cell RNA sequencing, flow cytometry, immunofluorescence, immunohistochemistry, and COMET analyses demonstrated that ProAgio plus chemotherapy reprogrammed the PDAC-TME by changing activated CAFs toward a qCAFs (quiescent CAFs), macrophages from protumor to proinflammatory polarization, and activating natural killer (NK) cells, CD4⁺, and CD8⁺ T cells. Combination therapy inhibited PDAC stemness. ProAgio-treated CAFs in vitro exhibited reduced secretion of glycine and cysteine, vital metabolites that support stemness and tumor progression. These results confirm the novel mechanism of action of ProAgio, which includes reducing hypoxia and modulating TME. The current data provide evidence for potentiation of chemotherapy efficacy by ProAgio.

This study aimed to investigate whether miR-425-5p contributes to post-spinal cord injury (SCI) inflammation and motor dysfunction by targeting CREB1. SCI rat models and H2O2-treated C8-D1A/C8-B4 cellular models were established. miR-425-5p and CREB1 were manipulated using inhibitors/antagomirs and siRNAs. Expression levels of miR-425-5p, IL-6, IL-1β, TNF-α, CREB1, and caspase-3 were measured using RT-qPCR. Cell apoptosis was evaluated by flow cytometry. Western blot analysis was performed to assess total CREB1 (t-CREB1) and phosphorylated CREB1 (p-CREB1) levels. Bioinformatics predictions were used to determine the targeting relationship between miR-425-5p and CREB1. BBB locomotor rating scale was employed to quantify motor function recovery in rats. miR-425-5p expression was markedly up-regulated in the H2O2-induced cell model, whereas CREB1 was down-regulated. CREB1 is a target of miR-425-5p. Inhibition of miR-425-5p significantly reduced apoptosis, suppressed pro-inflammatory cytokine expression, thereby promoting motor recovery; these effects were partially reversed by CREB1 knockdown. In SCI rats, miR-425-5p antagomir treatment alleviated inflammation and promoted motor function recovery; these beneficial effects were partially suppressed by co-administration of si-CREB1 to knockdown CREB1. MiR-425-5p upregulation in SCI directly suppresses CREB1 expression, subsequently exacerbating neuroinflammation, which in turn impairs functional recovery.

Etoposide is a widely used chemotherapeutic agent whose clinical application is limited by systemic toxicity and suboptimal intracellular delivery. Lipid-polymer hybrid nanoparticles have emerged as promising drug delivery systems that combine structural stability with enhanced cellular interaction. This study aimed to evalute the apoptotic and metastasis-related effects of etoposide-loaded lipid-polymer hybrid nanoparticles (ET-NPs) against human breast cancer cells. ET-NPs were synthesized and characterized, and their biological effects were compared with free etoposide in MCF-7 and MDA-MB-231 breast cancer cell lines. Cell viability was assessed using the MTT assay. Cell cycle distribution, Annexin V binding activity, mitochondrial membrane potential, and caspase activation were analyzed by flow cytometry based Muse Cell Analyzer. MMP-2 and MMP-9 mRNA levels were determined by quantitative real-time PCR. ET-NPs significantly enhanced the cytotoxic and pro-apoptotic effects of etoposide in both breast cancer cell lines. Annexin V analysis demonstrated increased apoptotic cell populations following ET-NP treatment compared with free etoposide. ET-NP also induced significant mitochondrial membrane depolarization, caspase activation, and G2/M phase arrest. Furthermore, ET-NP significantly exerted potential anti-metastatic activity indicating a preferential antitumor effect in MCF-7 cells compared with MDA-MB-231 cells. The findings of this study demonstrate that lipid-polymer hybrid nanoparticle-mediated delivery enhances the anticancer efficacy of etoposide in breast cancer cells by potentiating apoptotic signaling and suppressing metastasis-related gene expression. ET-NP may therefore represent a promising nanotherapeutic strategy for improving breast cancer treatment outcomes.

Inflammatory bowel disease (IBD) burden is rising globally, yet only subsets of patients benefit from available therapies, underscoring the need for more precise molecular and cellular stratification. In the PREDICT study, we enrolled treatment-naïve pediatric patients with IBD, alongside disorders of gut-brain interaction (DGBI) controls and healthy donors, and profiled their intestinal and blood-derived T cells using single-cell RNA sequencing (scRNA-seq). Across 107 participants, we identify a unique population of cytotoxic CD4⁺ T cells (CD4 CTL) enriched in the inflamed gut of patients with Crohn's disease (CD) and ulcerative colitis. CD4 CTLs are clonally expanded and express cytotoxic effector molecules and IFNG , consistent with antigen-driven activation. Cell-cell interaction analyses implicate macrophage-derived IL-27 as the top candidate for CD4 CTL differentiation, and IL-27 blockade in a mouse model limits CD4 CTL formation. Notably, elevated CD4 CTL frequencies in gut and peripheral blood at diagnosis are associated with subsequent poor outcome of anti-TNF therapy in pediatric CD. Findings in our identification cohort are validated in an independent cohort and through reanalysis of published datasets. Importantly, we designed a simple flow cytometry panel to isolate blood CD4 + CXCR6 + CD27 - T cells, which displayed a CD4 CTL transcriptional phenotype. Together, our results link CD4 CTLs to anti-TNF nonresponse and support their potential as an early, blood-accessible biomarker for treatment stratification in pediatric CD.

Intervertebral disc degeneration (IDD) serves as a predominant pathogenic driver of spinal degenerative diseases and low back pain. Long non-coding RNAs (lncRNAs) have emerged as important regulators involved in the pathogenesis of IDD. Despite this, the specific contributions of LINC00324, a particular lncRNA, to the advancement of IDD are not yet well understood. The study seeks to elucidate the functional role and explore the potential molecular mechanisms of LINC00324 in IDD. RT-qPCR was employed to analyze LINC00324, miR-143-3p, and TRAIL levels in intervertebral disc (IVD) tissues from IDD patients and control subjects, followed by correlation analysis. Bioinformatics tools, dual-luciferase reporter assays, and RNA pull-down experiments were conducted to validate the regulatory interactions within LINC00324/miR-143-3p/TRAIL in nucleus pulposus cells (NPCs). In vitro, NPCs were stimulated with lipopolysaccharide (LPS) to construct an IDD cell model. RT-qPCR, western blot, CCK-8 assay, and flow cytometry were utilized to evaluate the effects on the levels of LINC00324/miR-143-3p/TRAIL/DR4/DR5, as well as the proliferation and apoptosis of NPCs. Additionally, extracellular matrix (ECM)-related proteins and inflammatory factors were detected by western blot and ELISA, respectively. In vivo, a rat IDD model was established through fibroannular puncture. Radiological analysis (X-ray), HE staining, TUNEL staining, and western blot were used to assess disc structural changes, NPC apoptosis, and the levels of miR-143-3p/TRAIL/DR4/DR5 and apoptosis-related proteins. Clinical evidence showed that LINC00324 and TRAIL levels were increased, and miR-143-3p was decreased in IDD samples. miR-143-3p had the negative correlation coefficient with TRAIL and LINC00324. Furthermore, LINC00324 acted as a sponge for miR-143-3p, and miR-143-3p specifically attenuated the downstream of TRAIL level. In vitro, the LPS-induced IDD model showed decreased NPC viability, increased apoptosis, disrupted ECM metabolism (downregulated Collagen II and Aggrecan, upregulated MMP13), elevated inflammatory factors, and activated TRAIL/DR4/DR5 axis. Silencing LINC00324 or overexpressing miR-143-3p alleviated these pathological changes. Furthermore, miR-143-3p partially counteracted the promoting influences of LINC00324 overexpression on NPC apoptosis, ECM degradation, inflammatory response, and TRAIL/DR4/DR5 axis activation in the IDD model. In vivo, LINC00324 overexpression exacerbated IDD in rats (decreased disc height index, disrupted NPC tissue structure, enhanced NPC apoptosis, aggravated ECM degradation, inhibited miR-143-3p expression, and activated TRAIL/DR4/DR5 axis), while LINC00324 knockdown attenuated these degenerative changes. In conclusion, LINC00324 promotes IDD progression by inducing NPC apoptosis, disrupting ECM metabolism, and enhancing inflammatory response via sponging miR-143-3p to activate the TRAIL/DR4/DR5 axis. Not applicable.

Resistance training has been shown to activate the protein synthesis pathway, leading to muscle growth in humans. However, this type of exercise has shown equivocal results in animal studies due to the difficulty of mimicking muscle overload in vivo. This study aimed to determine whether ladder-based exercise in mice induces canonical molecular, cellular, and functional adaptations to training. Mice performed a single exercise session or 6 weeks of training in the ladder climb. Acute responses included mTOR phosphorylation, puromycin incorporation, and mRNA levels of myogenic regulatory factors (MRF). Chronic adaptations were assessed by strength, fat-free mass, physical performance, and blood lactate levels to confirm the training load. Sarcomeric proteins were analyzed using Western blot, while histology measured muscle fiber diameter and satellite cell (SC) fusion. The SC amount was quantified by flow cytometry. After a single exercise bout, mTOR phosphorylation increased at one and 3 h, with puromycin incorporation and MRF mRNA levels elevated at 8 h. After 6 weeks of training, the mice showed increased skeletal muscle strength and fat-free mass, with no changes in physical performance. Muscle-specific adaptations included increases in sarcomeric proteins and fiber diameters. SC adaptations were associated with an increased pool and enhanced capacity to fuse with muscle fibers. Our results demonstrate that ladder-based resistance exercise in mice induces molecular, cellular, and functional responses that are directionally consistent with adaptations reported after human resistance training, supporting its value for investigating the molecular and cellular mechanisms underlying this training.

Immune rejection after liver transplantation remains a major challenge impacting the long-term survival of liver transplant recipients. Inhibiting macrophage M1 polarization exerts a positive effect on alleviating post-transplant immune rejection. Arctiin, an active component derived from traditional Chinese medicine, may suppress macrophage M1 polarization by inhibiting the release of pro-inflammatory cytokines. This study employed experimental techniques including ELISA, double-label flow cytometry, Western blot analysis, and cellular immunofluorescence to investigate the potential mechanism underlying the effect of arctiin on macrophage M1/M2 phenotypic switching in vitro. Results demonstrated that arctiin promoted the conversion of M0 macrophages to the M2 phenotype, significantly downregulated the levels of pro-inflammatory cytokines IL-6 and TNF-α, inhibited the expression of the M1 macrophage marker inducible nitric oxide synthase (iNOS), and reduced the proportion of M1-type macrophages. Furthermore, arctiin notably enhanced STAT6 phosphorylation and TRAF6 protein expression in M1 macrophages while suppressing STAT1 phosphorylation. However, these beneficial effects of arctiin were significantly reversed by a CB2R antagonist. Additional experiments showed that a CB2R agonist exerted similar effects to arctiin. Treatment with a TRAF6 inhibitor abrogated the inhibitory effect of arctiin or the CB2R agonist on macrophage M1 polarization, increased STAT1 phosphorylation, and decreased STAT6 phosphorylation. In conclusion, this study indicates that arctiin can activate CB2R and exert anti-inflammatory effects through the TRAF6-STAT1/6 pathway, thereby promoting the phenotypic switch of macrophages from M1 to M2.

Extracellular vesicles (EVs) facilitate intercellular communication and reflect the physiological state of their parent cells. Microvesicles (MVs; ~200-900 nm) are released during cellular activation or stress and may provide insight into sex-related physiological differences. This study aimed to characterize circulating MV concentration, cellular origin, and MV microRNA cargo in healthy young males and females. Fasted sodium citrate plasma samples from healthy male (n = 16, age: 29 ± 6 years, BMI: 24.4 ± 2.4 kg/m2) and female (n = 16, age: 27 ± 5 years, BMI: 22.7 ± 1.9 kg/m2) participants were analyzed using size exclusion chromatography, tunable resistive pulse sensing, nano-flow cytometry, and RT-qPCR. MV concentration, antibody-defined MV subpopulations, and MV-miRNA cargo were assessed using linear models adjusted for age and BMI. Females exhibited higher concentrations of circulating platelet-derived CD41+ MVs (p = 0.03, d = 0.923), with a tendency for elevated monocyte-derived CD14+ MVs (p = 0.08, d = 0.546). No differences were observed for total MV concentration, remaining antibody-defined MV subpopulations, or MV-miRNA cargo expression. These findings demonstrate that healthy females exhibit elevated platelet- and monocyte-derived MV concentrations, while overall MV concentration and MV miRNA cargo are similar between sexes.

Streptococcus agalactiae infection remains a major constraint on the productivity of Nile tilapia (Oreochromis niloticus), a species of considerable economic importance in global aquaculture. Although selective breeding programs have successfully produced tilapia strains with enhanced resistance to this pathogen, the B cell-mediated immune mechanisms underlying this resistance remain unclear. In this study, we systematically investigated differences in IgM+ B cell mediated immune responses between S. agalactiae-resistant and susceptible Nile tilapia. Transcriptomic analysis of splenic leukocytes revealed that genes associated with immunoglobulin production and B cell differentiation were markedly upregulated in resistant tilapia following infection. Consistent with these findings, flow cytometry further demonstrated a more pronounced expansion of splenic IgM+ B cells in resistant tilapia, which was primarily driven by significantly increased proliferative activity. Importantly, this expansion coincided with elevated activation induced apoptosis, suggesting an accelerated B cell turnover that could facilitate the selection and maintenance of functionally competent cells during the immune response. Functionally, IgM+ B cells from resistant tilapia exhibited sustained enhancement of antigen processing capacity following infection. Meanwhile, macrophage-mediated phagocytosis of S. agalactiae increased progressively and became particularly evident at later stages of infection. Moreover, IgM+ B cells in resistant tilapia maintained relatively stable intracellular ROS levels, whereas susceptible tilapia exhibited persistent oxidative stress. Collectively, these findings indicate that resistance to S. agalactiae infection in Nile tilapia is closely associated with coordinated functional remodeling of IgM+ B cells, characterized by efficient proliferation, functional maturation, and stringent homeostatic regulation. This refined B cell-mediated immunity likely contributes to the effective pathogen clearance and provides an immunological basis for the evaluation and selective breeding of disease-resistant tilapia strains.

The clinical significance of PNH Type II white blood cells (WBCs) remains unclear. We assessed the relative percentage (rel%) of Type II neutrophils in 355 patients with a PNH clone ≥ 1% on neutrophils enrolled by 33 flow cytometry laboratories in the 5-year French nation-wide multicenter prospective observational study. We first analyzed 127 of 133 patients with a major PNH clone (≥ 50%) and evaluable Type II neutrophil rel%. Using hemolysis data available for 107 patients, a threshold of 3% Type II neutrophils rel% distinguished two groups: 22 "high Type II" and 105 "low Type II" patients. Hemolysis was less frequent in the "high Type II" group. Thrombosis was more frequent at diagnosis and during cumulative long-term follow-up in this group. High neutrophil rel% ≥ 3% was independently associated with thrombotic events. Among 222 patients with smaller PNH clones (1%-50%), 207 displayed evaluable Type II neutrophil rel%, of which 87 were classified as "high Type II" and 120 as "low Type II". Although no thrombosis was present at diagnosis, two thrombotic events occurred during follow-up in the "high Type II" group, whereas none were observed in the "low Type II" group. Long-term follow-up showed no major changes in Type II neutrophil rel%, irrespective of variations in total clone size. These findings suggest that, regardless of the total PNH clone size, PNH Type II neutrophil rel% is a stable parameter and may represent a reliable predictor of thrombosis, potentially supporting earlier consideration of anticomplement therapy initiation.

Tumor necrosis factor-α-stimulated gene-6(TSG-6), a secreted protein with anti-inflammatory and tissue-protective properties, mediates a cascade of proinflammatory cytokines and ameliorates tissue fibrosis. Previous studies have found that TSG-6 can attenuate the degree of fibrosis, inhibit the inflammatory response, and reduce adipogenesis in orbital tissues in a Thyroid Eye Disease (TED) mouse model of thyroid-eye disease; however, the exact mechanism has not been elucidated. In the present study, we investigated the mechanism by which TSG-6 exerts its anti-inflammatory and antifibrotic effects in an in vitro cellular model of TED. Human orbital connective tissue was collected from primary and passaged cultures and 3-5 passages-cells were used in subsequent experiments. The expression of relevant inflammatory markers, including tumor necrosis factor-α(TNF-α), Interleukin-6(IL-6), monocyte chemoattractant protein-1(MCP-1), Cyclooxygenase-2(COX-2), and intercellular cell adhesion molecule-1(ICAM-1), was detected by western blotting with 5, 10, and 15 ng/ml TSG-6 pretreatment in the presence or absence of 10 ng/ml Interleukin-1beta (IL-1β) and H2DCFDA (DCFH-DA) fluorescent staining, and flow cytometry was used to detect reactive oxygen species(ROS) indices. Orbital fibroblasts (OF) were treated with TSG-6 in the presence or absence of transforming growth factor-beta 1(TGF-β1) agonist (SRI-011381:MCE, HY-100347) and the expression of TGF-β1/Smad pathway-associated fibrosis factors, including α-smooth muscle actin (α-SMA), connective tissue growth factor (CTGF), and collagen type 1 (COL1A1). TSG-6 inhibited IL-1β-induced production of the inflammatory mediators TNF-α, IL-6, MCP-1, COX-2, and ICAM-1, and the release of ROS in a dose-dependent manner in an in vitro cell model of TED. TSG-6 downregulated the expression of TGF-β1, Smad2/3, p-smad2/3, α-SMA, CTGF, and COL1A1 in a dose-dependent manner after TGF-β1 pretreatment and reduced the phosphorylation of Smad2/3, suggesting that TSG-6 inhibits the TGF-β1 signaling cascade response in orbital fibroblasts. TSG-6 inhibited the production of inflammatory mediators and the release of ROS, and suppressed fibrosis of human orbital fibroblasts by downregulating the TGF-β1/Smad pathway. These data suggest a potential application of TSG-6 in the treatment of TED and provide a novel target for the treatment of TED.

Copper-containing laccases oxidize various phenolic and non-phenolic compounds. Among them, fungal laccases have been extensively studied in industry, but their biomedical potential remains largely unexplored. We aimed to investigate the anticancer activity of the laccase enzyme, which we purified from Trametes versicolor without loss of activity using the three-phase partitioning (TPP) technique-an easy, inexpensive method that allows high activity recovery-on colorectal cancer (CRC) cells for the first time and to elucidate its mechanism of action. The effects of the pure laccase enzyme were investigated on two CRC cell lines (HCT-116 and Caco-2) and normal endothelial cells (HUVEC). Cytotoxicity was assessed using the WST-1 assay. Apoptosis and cell cycle progression were analyzed by flow cytometry. Genotoxicity was determined by using micronucleus and comet assays. Intracellular DCFH-DA fluorescence intensity was measured, and the expression of apoptosis- and DNA repair-related genes was analyzed by qRT-PCR. The MAPK (p38, JNK) and NF-κB signaling pathways were examined by immunofluorescence. Laccase treatment exhibited a dose-dependent cytotoxic effect on CRC cells, with minimal impact on HUVECs. It induced apoptosis, oxidative stress-associated responses, DNA damage, and G0/G1 cell cycle arrest. Gene expression analysis revealed upregulation of Bax and Caspase-3 and downregulation of Bcl-2, RAD51, and NF-κB. Immunofluorescence confirmed activation of p38/JNK and suppression of NF-κB signaling. TPP-purified fungal laccase exhibits selective cytotoxicity and pro-apoptotic effects in CRC cells via redox-mediated DNA damage and modulation of signaling pathways, supporting its potential as a novel anticancer agent.

Combination therapy integrating bispecific antibody (BsAb) and oncolytic viruses (OVs) to enhance antitumor immune response offers a promising therapeutic approach in comparison with OV treatment alone. This study aims to strengthen and characterize NK cell-mediated antitumor responses using modified oncolytic viruses, i.e., ONCOS-102 and ONCOS-204 genetically engineered to express Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) and the ligand of inducible T-cell co-stimulator (ICOSL), respectively, in combination with a BsAb targeting CD16 and the epidermal growth factor receptor (EGFR). Changes in phenotype, degranulation, cytokine production, and cytotoxicity of NK cells induced by combined treatment were compared with those induced by individual treatment strategies against non-small cell lung cancer, malignant melanoma, and ovarian cancer. Using flow cytometry and colorimetry-based cytotoxic assays, our results showed that EGFRxCD16 BsAb was the main variable in driving NK cells toward an activated phenotype and enhanced NK function; while the OVs alone did not demonstrate drastic impact on NK cells. Interestingly, tumor preconditioning with OVs in combination with EGFRxCD16 BsAb showed the most potent NK cytotoxicity in comparison with EGFRxCD16 BsAb alone and appeared to synergize best against ovarian and EGFRmut lung tumor cell lines. Despite differences between tumor types, our data suggest a favorable interplay between OVs, especially ONCOS-102, and EGFRxCD16 BsAb  in sensitizing NK cell antitumor response in vitro. These results warrant further in vivo exploration and clinical translation of this promising combination of therapeutic modalities.

Tumor vaccines largely depend on targetable tumor-specific neoantigens. However, in certain tumors, mutation-based neoantigens are exceptionally rare or even absent. To address this challenge, we developed an innovative strategy beyond genomic mutation events by utilizing tumor-specific chimeric RNAs and their encoded chimeric proteins. In this study, we demonstrated through flow cytometry and cell counting kit-8 (CCK-8) assays that aberrantly spliced tumor-specific chimeric RNAs (e.g., the ASTN2-PAPPA antisense chimeric RNA, A-PaschiRNA) can serve as tumor neoantigens. Furthermore, utilizing mouse tumorigenesis models, we have developed a novel therapeutic strategy involving extracellular vesicle (EV)-based vaccines loaded with chimeric RNAs to treat esophageal squamous cell carcinoma.

Hepatotoxicity is a common and significant adverse effect associated with adeno-associated virus (AAV) gene therapy; however, the underlying mechanisms remain unknown. In this study, we demonstrated innate immune activation in two patients with spinal muscular atrophy shortly after receiving AAV9 gene therapy with onasemnogene abeparvovec. Both patients developed marked hyperferritinemia accompanied by hepatotoxicity, thrombocytopenia, hypertriglyceridemia, and hypofibrinogenemia, all of which are the diagnostic criteria for macrophage activation syndrome. To evaluate their immune responses, serial analyses of serum cytokines/chemokines and flow cytometry were performed. Surges in macrophage-associated cytokine levels were observed in proportion to the severity of adverse events within 1 week after AAV vector infusion, suggesting that macrophage activation contributed to the pathogenesis of these adverse effects. Our findings clarify the immunological basis underlying hepatotoxicity in patients after AAV gene therapy. Furthermore, these findings provide a rationale for using various immunosuppressants or chemokine blockers in patients exhibiting severe adverse effects.

Heritable platelet function disorders (HPFD) are a diverse group of bleeding disorders characterised by a primary qualitative defect in platelet function rather than platelet number. HPFD may be broadly categorised according to the severity of bleeding, with Glanzmann thrombasthenia and Bernard Soulier syndrome classically considered severe HPFD, with well-described clinical and laboratory phenotypes. The remaining non-severe HPFD are less completely characterised and provide additional challenges in diagnosis despite advances in standardising diagnostic approaches for HPFD. Distinguishing patients with non-severe HPFD from patients who do not have a bleeding disorder may be aided by employing bleeding assessment tools early in patient assessment, although there may be limitations according to patient factors. Distinguishing patients with non-severe HPFD from those with acquired platelet function disorders and from those with other heritable disorders of haemostasis (including bleeding disorder of unknown cause) requires more sophisticated clinical and laboratory tools, including platelet light transmission aggregometry, secretion and flow cytometry assays, which can categorise many HPFD to the level of the defective platelet pathway and some to the level of an individual protein. The increasing availability of high-throughput genetic sequencing has made genetic diagnosis possible for some patients with non-severe HPFD; however, this is limited by difficulties with standardisation of laboratory phenotyping and the challenges of distinguishing pathogenic from benign genetic variation. This review will focus on updates in diagnostic approaches for patients with non-severe HPFD since the last IJLH education article on this topic in 2014.