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Lipid nanoparticles (LNPs) are small particles composed of lipids that can be used to encapsulate and deliver therapeutic substances like mRNA or other drugs to specific cells or tissues. LNPs are specifically designed to protect the payload from degradation, enhance payload stability, and improve delivery to target cells and tissues. When developing LNPs for nucleic acid-based therapeutics, there are two characteristics that are often analyzed to determine the quality of the formulations: size distribution and loading efficiency. Various techniques are used to determine the size of nanoparticles, with each providing different details and having specific advantages and limitations. In this study, we evaluated the use of nanoparticle tracking analysis, dynamic light scattering, and two different nano cytometers to determine LNP size. We compared the results obtained from these four methods to the particle size determined using cryogenic transmission electron microscopy. Our findings showed that nano-flow cytometry instrumentation, like NanoFCM and CytoFLEX nano, offers a robust and accurate method for sizing LNPs with measurements in high concordance to those obtained by conventional cryogenic transmission electron microscopy.
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.
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.
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.
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.
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.
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.
Polyneuropathy due to antibodies to myelin-associated glycoprotein (MAG) is a rare disease with an estimated prevalence of 1 per 100,000. Symptoms start in the sixth or seventh decade of life, presenting sensory polyneuropathy with sensory ataxia, paresthesia, mild motor deficit, and tremor of upper extremities. The neurophysiological features are compatible with length-dependent demyelination. It is caused by monoclonal gammopathy of the IgM type produced by clonal B cells. In recent years the monoclonal anti-CD20 antibody has become the preferred first-line treatment, but with limited efficacy. We identified a cohort of 42 patients diagnosed with anti-MAG polyneuropathy. Clinical data were collected retrospectively, and flow cytometry files and immune histochemistry slides were reassessed. We report that in most cases of anti-MAG polyneuropathy, the monoclonal IgM is kappa-restricted, the B-cell population shows lymphoplasmacytic differentiation, MYD88L265P mutation is a frequent finding, and the clonal B-cell population includes plasma cells. These findings most likely explain the low efficacy of rituximab monotherapy. We found the burden of symptoms to be high among patients with anti-MAG polyneuropathy as most of our patients had received intravenous immunoglobulin infusions and/or B-cell-directed treatment. We suggest that future treatment protocols for anti-MAG polyneuropathy should incorporate plasma cell-directed drugs. Furthermore, we found complement receptor 1 (CD35) to be down regulated on clonal B lymphocytes but not on normal B lymphocytes in these patients. We suggest that this may be a common feature of clonal B lymphocytes in chronic lymphoproliferative diseases.
Anlotinib, a novel multi-target tyrosine kinase inhibitor, has demonstrated promising antitumor efficacy by inhibiting angiogenesis. However, the potential therapeutic benefits and underlying mechanisms of combining anlotinib with immune checkpoint inhibitors in colorectal cancer remain unclear. Syngeneic models of colorectal cancer were established and treated with anlotinib, PD-1/PD-L1-IN-9 hydrochloride, or their combination. Tumor tissues were analyzed using immunohistochemistry, and Western blotting to assess angiogenesis-associated markers, tissue hypoxia, and key molecular markers. Flow cytometry, ELISA, and immunostaining were performed to evaluate immune cell infiltration, cytokine expression, and the tumor immune microenvironment. The combination of anlotinib and PD-1/PD-L1-IN-9 hydrochloride significantly inhibited tumor growth compared to monotherapy, associated with improved neovascularization and alleviated hypoxia. Combined therapy increased CD8+ T cell infiltration, reduced immunosuppressive cell populations, and partially modulated cytokine profiles, thereby enhancing the antitumor immune response. Anlotinib combined with PD-1/PD-L1-IN-9 hydrochloride exhibits superior antitumor efficacy in colorectal cancer compared to either agent alone, potentially by reshaping the tumor microenvironment. These findings support further exploration of combined anti-angiogenic and immune checkpoint therapy as a promising strategy for colorectal cancer treatment.
In this study, we have a type of functional nanofibers, hybrid nanofibers containing Polycaprolactone (PCL) / Chitosan (CS) and Polyvinyl alcohol (PVA) were successfully fabricated by hybrid electrospinning method. Pioglitazone Hydrochloride (PIO) was incorporated in PCL/CS solution due to its hydrophobic nature, and Gemcitabine Hydrochloride (GEM) was incorporated in PVA solution due to its hydrophilic nature. The prepared hybrid PCL-CS-PIO/PVA-GEM nanofibers exhibited a biphasic release profile in each case, indicating an initial burst followed by a sustained phase of GEM and PIO over 5 days. The maximum amount released from the hybrid nanofibers reached 75.88 ± 1.5% of the GEM and 64.54 ± 2.05% of the PIO in a medium of pH 7.4. Flow cytometry and MTT analyses demonstrated that the combination of PIO and GEM induced apoptosis and inhibited the growth of melanoma cancer cell lines by inhibiting proliferation with cell viability of 51.34 ± 2.2% and 46.04 ± 1.5% after 24 and 48 h compared to control group. Obtained nanofibers were assessed for their chemical composition and morphology followed by scanning electron microscopy (SEM), FTIR, contact angle measurements and drug release profile. Then, a cytotoxicity study was performed on human melanoma cancer cell line, followed by investigating its potential in treatment applications.
The complement system is crucial in antineutrophil cytoplasmic antibody-associated vasculitis (AAV) pathogenesis. Extracellular vesicles (EVs) serve as carriers of bioactive substances, influencing the immune system. We aimed to investigate myeloperoxidase-positive EVs (MPO+EVs) exposing complement components (C3a, C4d), terminal complement complex (TCC) and complement factor B (CFB) in relation to disease activity and kidney involvement. Plasma MPO+EVs carrying complement products, C3a, C4d, TCC or CFB, were analysed by flow cytometry. Clinical data, including kidney biopsy findings, were retrieved. Disease activity was assessed using the Birmingham Vasculitis Activity Score (BVAS). Eighty-one patients with AAV with granulomatosis with polyangiitis (n=59) or microscopic polyangiitis (n=22) were included. Active disease was noted in 73 (90.1%) patients and 50 (68.5%) had kidney involvement. Patients with kidney involvement had higher concentrations of MPO+, MPO+C3a+, MPO+C4d+ and MPO+TCC+EVs compared to patients without, and concentrations correlated positively with BVAS (p<0.05), respectively. Levels of EVs showed a negative correlation with estimated glomerular filtration rate, and a positive correlation with the proportions of necrosis and crescents in kidney biopsies. ROC curve analysis indicated that MPO+EVs could distinguish patients with kidney involvement from non-renal AAV. A binary multivariable logistic regression confirmed an independent association between levels of EV subsets and kidney involvement. Elevated levels of MPO+EVs exposing complement components in patients with kidney involvement indicate activation of the classical or lectin and common complement pathways in renal AAV. Furthermore, the association between levels of MPO+EVs with the presence of crescents and necrotic lesions in kidney tissue supports a role in renal inflammatory activity.
The role of psychological factors in the holistic regulation of cancer has garnered significant attention. Psychological eustress induced by an enriched environment (EE) inhibits various cancers including melanoma, but underlying mechanisms remain largely unknown. The brain-gut axis may play a key role in modulating psychological factors and cancer progression. This study investigates the role of gut microbiota in EE-induced anti-melanoma effects. C57BL/6 mice were housed in EE or standard environment (SE). Longitudinal fecal samples from EE and SE mice underwent 16S rRNA gene sequencing. SE melanoma-bearing mice received fecal microbiota transplantation (FMT) from EE or SE donors to assess the microbiota's causal role in EE's anti-tumor effects. Bacterial species upregulated by EE in tumor-bearing mice were identified;Parabacteroides distasonis (Pd) was selected for therapeutic administration. Immune cells in spleen and tumor were quantified by flow cytometry. Natural killer (NK) cell function was tested using anti-NK1.1 depletion. EE increased the alpha diversity of the gut microbiota and alleviated the dysbiosis caused by melanoma. FMT from EE mice to SE mice inhibited melanoma growth, suggesting that the gut microbiota contributes to EE's anti-tumor effects. EE upregulated seven bacterial species in tumor-bearing mice, including Pd. Oral administration of Pd inhibited melanoma growth and increased intratumoral NK/NKT cell proportions and NK cell granzyme B expression. NK cell depletion abrogated Pd's anti-tumor effect. This study underscores the interconnectedness of psychological eustress, gut microbiota, and cancer, providing preclinical insights into holistic cancer treatments. Leveraging the brain-gut-cancer axis, targeting psychological factors and gut microbiota could offer a potential adjunctive strategy for melanoma management.
Gestational diabetes mellitus (GDM) is a common metabolic disorder of pregnancy associated with placental dysfunction and adverse maternal-fetal outcomes. LncRNAs have emerged as important regulators in metabolic diseases. The clinical significance and molecular mechanisms of lncRNA PANDAR in GDM remain unclear. 117 GDM patients and 110 healthy pregnant women were enrolled. Relative expression of genes was measured by qRT-PCR. The diagnostic value of PANDAR was assessed using ROC analysis, and its association with adverse pregnancy outcomes was evaluated by logistic regression. High-glucose model was established in HTR-8/Svneo trophoblast cells to investigate the function of PANDAR. Cell apoptosis, viability, and invasion were assessed using flow cytometry, CCK-8, and Transwell assays. The regulatory relationships among molecules were validated by dual-luciferase reporter assays. PANDAR expression was upregulated in GDM patients and exhibited good diagnostic performance. PANDAR levels were higher in GDM patients with adverse pregnancy outcomes. Elevated PANDAR was independently associated factor for adverse pregnancy outcomes. High glucose induced PANDAR expression and promoted trophoblast apoptosis while inhibiting cell viability and invasion, effects that were alleviated by PANDAR silencing. PANDAR functioned as a ceRNA for miR-192-5p, thereby upregulating the pro-apoptotic target BCL2L11. Rescue experiments confirmed that a PANDAR/miR-192-5p/BCL2L11 axis mediated trophoblast dysfunction under hyperglycemic conditions. PANDAR is associated with adverse pregnancy outcomes and may reflect hyperglycemia-induced cellular responses by promoting trophoblast dysfunction via a miR-192-5p/BCL2L11 axis.
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.
Inhibitory receptor signalling represents a potential therapeutic strategy in rheumatoid arthritis (RA), but its effects across pathogenic CD4⁺ T cell subsets and T-B cell interactions remain incompletely defined. We aimed to delineate the expression and function of major inhibitory receptors, including PD-1, BTLA, and TIGIT, in RA synovial CD4⁺ T cells and to determine how receptor agonism modulates T cell effector functions and T-B cell interactions. CD4⁺ T cells were isolated from RA synovial fluid or from tonsils and blood of controls. Cells were cocultured with artificial antigen-presenting cells (murine fibroblast L cells) expressing PD-L2, HVEM, or CD155, and cytokine production was measured by ELISA (Enzyme-Linked Immunosorbent Assay). Three-dimensional organoid cultures were employed to assess PD-L2-mediated regulation of age-associated B cell (ABC) induction by flow cytometry. Single-cell RNA sequencing profiled inhibitory receptor and ligand expression across T cell subsets. PD-1, BTLA, and TIGIT were preferentially expressed on T peripheral helper (Tph) and T follicular helper (Tfh) cells in RA synovium. Engagement of PD-L2 broadly suppressed pathogenic cytokine production across multiple CD4⁺ T cell subsets, including IL(Interleukin)-21 and CXCL13 in Tph cells and TNF (Tumor Necrosis Factor), IFN-γ (Interferon-γ), and IL-2 in PD-1⁺CXCR5⁻ non-Tph cells, with effects abolished by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)-mediated PD-1 disruption. PD-L2 also inhibited cytokine production by tonsillar Tfh and non-Tfh cells. In 3-dimensional organoids, PD-L2 significantly reduced the induction of CD11c⁺CD21⁻T-bet⁺ ABCs. PD-1 activation suppresses multiple T cell populations from diverse sites, including Tph cells in RA synovium. These results support the broad potential of PD-1 agonism to suppress pathologic T cell responses in RA.
Hepatocellular carcinoma (HCC) is a leading cause of cancer-related death worldwide. Targeting cancer cells using functionalized nanoparticles has gained attention. In this study, the anticancer potential of Iron oxide NPs functionalized with Glucose and co-conjugated with Hyaluronic acid and trans-Chalcone (Fe3O4@Glu-HA-TC NPs) and their influence on the expression of the lncRNAs ANRIL and ANCR was investigated. The synthesized NPs were characterized by FT-IR, XRD, SEM, EDS, DLS, zeta potential, TGA and VSM analysis. Viability level of the HepG2 and HDF cell lines was studied by MTT assay and cell cycle phases and apoptosis/necrosis percentage and ROS levels in the HepG2 cell treated with Fe3O4@Glu-HA-TC NPs were determined. Relative expression of the lncRNAs ANCR and ANRIL was determined by Real-Time PCR assay. The NPs were spherical, moderately agglomerated, with an average particle diameter of 42.05 nm, surface charge of -56.8 mV, hydrodynamic size of 150.9 nm and saturated magnetization at 38.88 emu/g. Fe₃O₄@Glu-HA-TC NPs exhibited greater cytotoxicity toward HepG2 cells compared to normal HDF cells with an IC50 of 255 and 488.79 µg/mL, respectively. Exposure to the NPs caused an apparent blockage at the sub-G1 phase, notably elevated early and late apoptosis (as observed in a representative flow cytometry experiment), and induced ROS generation by approximately 3.4-fold. In addition, treatment with Fe3O4@Glu-HA-TC NPs significantly upregulated the lncRNA ANCR (1.52-fold) while slight downregulation the ANRIL (0.89-fold). The present work provides evidence that Fe₃O₄@Glu-HA-TC NPs induce cytotoxicity, cell cycle arrest, and apoptosis in HepG2 cells, with associated ROS generation and lncRNA expression changes. However, mechanistic validation (e.g., antioxidant rescue, lncRNA functional assays) is needed to establish causality. It should be noted that while hyaluronic acid functionalization was employed as a rationale for potential CD44-mediated uptake, direct evidence of receptor-mediated targeting was not obtained in this study.
Circular_RNAs (circ_RNAs) are involved in the development and progression of human malignancies, including breast cancer. Novel circ_RNAs for breast cancer remain to be further determined. This study investigates the role and mechanism of a novel circ_RNA circ_0048766 in triple-negative breast cancer (TNBC) progression. Bioinformatics analysis was performed using the GSE165884 database to identify differentially expressed circ_RNAs. The expression of circ_0048766 was validated in various breast cancer cell lines through quantitative real-time PCR. Functional assays, including CCK-8, flow cytometry, colony formation, and Transwell assays, were conducted in BT-549 and MDA-MB-231 cells. The mechanisms involving methyltransferase-like 3 (METTL3), miR-329-3p, and C-X-C Motif Chemokine Receptor 4 (CXCR4) were examined through bioinformatics, dual-luciferase reporter assays, RNA immunoprecipitation, and RNA pull-down. In vivo studies were conducted using a nude mouse xenograft model to evaluate tumor growth and CXCR4 expression. Circ_0048766 was significantly upregulated in TNBC cell lines compared to normal breast epithelial cells. Knockdown of circ_0048766 resulted in reduced cell viability, colony formation, migration, and invasion while increasing apoptosis in BT-549 and MDA-MB-231 cells. Mechanistically, circ_0048766 was confirmed to function as a sponge for miR-329-3p. Moreover, miR-329-3p directly suppressed CXCR4 expression, and circ_0048766 regulated CXCR4 in a miR-329-3p-dependent manner. Additionally, circ_0048766 promoted epithelial-mesenchymal transition, as evidenced by its regulation of E-cadherin, N-cadherin, and Vimentin expression via miR-329-3p. MiR-329-3p inhibition or CXCR4 overexpression reversed the effects of circ_0048766 knockdown. In vivo, circ_0048766 knockdown significantly diminished tumor growth and weight, along with decreased CXCR4 levels. METTL3 was identified as an upstream regulator mediating m6A modification of circ_0048766. Functionally, METTL3 knockdown suppressed CXCR4 expression and cell proliferation, effects that were rescued by CXCR4 overexpression. Circ_0048766 is a novel oncogenic circ_RNA that promotes TNBC progression through the miR-329-3p/CXCR4 signaling axis, regulated by METTL3. This study highlights the potential of targeting the METTL3/circ_0048766/miR-329-3p/CXCR4 axis as a novel therapeutic strategy for TNBC treatment. Not applicable.
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.
Angiotensin (Ang) II can cause podocyte injury and leads to chronic kidney disease (CKD), but the specific mechanism is not clear. In our previous study, we found that Ang II reduced the expression of cholesterol efflux associated molecule by regulating Sirtuin 6 (SIRT6), thereby inducing podocyte cholesterol accumulation and injury. However, the effect of SIRT6 on podocyte cholesterol synthesis is unclear. In this study, we evaluated the role of SIRT6 in the expression of cholesterol synthesis associated molecular sterol regulatory element-binding protein 2 (SREBP2), cholesterol content, and apoptosis levels in podocytes. Ang II-infused rat model was constructed. Cholesterol quantification was examined using a cholesterol quantitation kit. The expression of SIRT6 and SREBP2 were examined by western blot and immunofluorescence assay. The podocyte apoptotic level was detected by flow cytometry. Transfection of podocytes with pcDNA 3.1 SIRT6 plasmid and the deacetylase-inactive SIRT6 mutant plasmid (pcDNA 3.1 SIRT6 H133Y) was performed to investigate whether SIRT6 regulates SREBP2 expression and to further determine if this regulatory effect is dependent on SIRT6's deacetylase activity. Ang II promoted lipid droplet and cholesterol accumulation in rat glomeruli, suppressed SIRT6 expression, and enhanced SREBP2 activation, evidenced by increased SREBP2 levels and nuclear translocation. Transfection of pcDNA3.1SIRT6 into podocytes attenuated SREBP2 activation, reducing Ang II-induced cholesterol accumulation and apoptosis. Experiments using pcDNA3.1 SIRT6 and pcDNA3.1 SIRT6 H133Y plasmids indicate that SIRT6-mediated suppression of SREBP2 requires its histone deacetylase activity. We conclude that SIRT6 serves as a key protective regulator of podocytes against RAS activation, at least in part by suppressing SREBP2 expression and thereby alleviating cholesterol accumulation and associated podocyte injury. It provides a new target for clinical treatment of CKD.
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.