Search Results (131)

Background: Melanoma metastasis, driven by tumor microenvironment (TME)-mediated crosstalk facilitated by extracellular vesicles (EVs), remains a major therapeutic challenge. A critical barrier to clinical translation is the overlap in protein cargo between tumor-derived and healthy cell EVs. Objective: To address this, we developed Scaffold-free Functional Deconvolution (SFD), a novel computational approach that leverages a comprehensive healthy cell EV protein database to deconvolute non-oncogenic background signals. Methods: Beginning with 1915 proteins (identified by MS/MS analysis on an Orbitrap Fusion Lumos Mass Spectrometer using the IonStar workflow) from melanoma EVs isolated using REIUS, SFD applies four sequential filters: exclusion of normal melanocyte EV proteins, prioritization of metastasis-linked entries (HCMDB), refinement via melanocyte-specific databases, and validation against TCGA survival data. Results: This workflow identified 21 high-confidence targets implicated in metabolic-associated acidification, immune modulation, and oncogenesis, and were analyzed for reduced disease-free and overall survival. SFD’s versatility was further demonstrated by surfaceome profiling, confirming enrichment of H7-B3 (CD276), ICAM1, and MIC-1 (GDF-15) in metastatic melanoma EV via Western blot and flow cytometry. Meta-analysis using Vesiclepedia and STRING categorized these targets into metabolic, immune, and oncogenic drivers, revealing a dense interaction network. Conclusions: Our results highlight SFD as a powerful tool for identifying clinically relevant biomarkers and therapeutic targets within melanoma EVs, with potential applications in drug development and personalized medicine. Full article

Compelling clinical evidence strongly indicates that anti-angiogenesis therapeutics including Bevacizumab, a humanised anti-VEGF mAb, can alleviate the resistance to immunotherapy. We explored the direct modulation of Bevacizumab on endothelial cell (EC) immune response including surface expression of adhesion and MHC molecules and EC-elicited proliferation of immune cells under inflammatory conditions. Flow cytometry showed that addition of VEGF inhibited TNF-α stimulation of expression of ICAM-1 and VCAM-1 on HUVECs, whereas Bevacizumab enhanced this TNF-α-stimulated expression. The presence of MHC Class I on HUVECs was decreased by VEGF and increased by TNF-α, respectively. Bevacizumab reversed VEGF downregulation and promoted TNF-α upregulation of MHC class I expression, suggesting that anti-VEGF treatment can boost the endothelial immunological reaction, a prerequisite for immune cell trafficking. Functionally, real-time monitoring of the proliferation of human PBMCs co-cultured on HUVEC monolayers over 3 days showed opposing effects on the proliferation of PBMCs between VEGF and TNF-α. Consistently, Bevacizumab antagonised VEGF suppression and sensitized TNF-α activation of PBMC growth over the time course. In line with these findings, Bevacizumab increased the surface expression of CD69 on VEGF-treated T cells collected from PBMCs after 3-day co-cultures with HUVECs. Furthermore, the proliferation of CD3+, CD8+ and CD4+ T cells was promoted via Bevacizumab. Collectively, this study demonstrates that targeting VEGF can enhance the immune response of ECs required for T cell recruitment. Our findings provide insights to a deeper understanding of increased vascular inflammatory response conferred by anti-VEGF treatment in addition to inhibiting angiogenesis, which supports its favourable dual role in the positive immunological synergism with immunotherapy. Full article

Currently, there are two major theories regarding the pathogenesis of sepsis: hyperimmune and hypoimmune. The hyperimmune theory suggests that a cytokine storm causes the symptoms of sepsis. On the contrary, the hypoimmune theory suggests that immunosuppression causes the manifestations of sepsis. By conducting a microarray analysis on peripheral leukocytes from patients with sepsis, this study found that hyperactivity of TH17 immunity was noted in sepsis patients. Innate immunity-related genes are significantly upregulated, including CD14, TLR1,2,4,5,8, HSP70, CEBP proteins, AP1 (JUNB and FOSL2), TGFB1, IL6, TGFA, CSF2 receptor, TNFRSF1A, S100A binding proteins, CCR2, FPR2, amyloid proteins, pentraxin, defensins, CLEC5A, whole complement machinery, CPD, NCF, MMP, neutrophil elastase, caspases, IgG and IgA Fc receptors (CD64, CD32), ALOX5, PTGS, LTB4R, LTA4H, and ICAM1. The majority of adaptive immunity genes were downregulated, including MHC-related genes, TCR genes, granzymes/perforin, CD40, CD8, CD3, TCR signaling, BCR signaling, T and B cell-specific transcription factors, NK killer receptors, and TH17 helper-specific transcription factors (STAT3, RORA, and REL), as well as Treg-related genes, including TGFB1, IL15, STAT5B, SMAD2/4, CD36, and thrombospondin. The findings of this study show that Th17 with Treg over-presentation play an important role in the pathophysiology of sepsis. Full article

Despite the progress in cancer immunotherapy, therapeutic responses in solid tumors remain suboptimal due to the immunosuppressive nature of the tumor microenvironment (TME), limited immune cell infiltration, and inefficient delivery of immune-activating agents. Dendritic cell-based therapies possess strong immunological potential but face challenges in viability, standardization, and scalability. Likewise, exosomes and CAR-T cells are hindered by instability, production complexity, and limited efficacy in immune-excluded tumor settings. Objective: This study evaluates dendritic cell-derived vesicles (DC-Vesicles), embedded in a phospholipid-rich structural scaffold, as a multi-functional and scalable platform for immune modulation and therapeutic delivery. We aimed to assess their structural stability, immune marker preservation under clinical processing conditions, and potential to reprogram the TME. Methods and Results: DC-Vesicles were generated and analyzed using bottom-up proteomics via nanoLC–MS/MS on a timsTOF Pro 2 system under three conditions: fresh, concentrated, and cryopreserved. A consistent proteomic profile of over 400 proteins was identified, with cryopreserved samples retaining >90% of immune-relevant markers. Differential expression analysis confirmed stability of key immunological proteins such as HLA-A, QSOX1, ICAM1, NAMPT, TIGAR, and Galectin-9. No significant degradation was observed post-cryopreservation. Visualization through heatmaps, PCA, and volcano plots supported inter-condition consistency. In silico modeling suggested preserved capacity for M1 macrophage polarization and CD8⁺ T cell activation. Conclusions: DC-Vesicles demonstrate structural resilience and functional retention across storage conditions. Their cold-chain-independent compatibility, immune-targeting profile, and potential regulatory classification as Non-New Chemical Entities (NCEs) support their advancement as candidates for precision immunotherapy in resistant solid tumors. Full article

CD4⁺ T cells have been well-regarded as “helper” cells in activating the cytotoxicity of CD8⁺ T cells for effective tumor eradication, while few studies have focused on whether CD8⁺ T cells regulate CD4⁺ T cells. Our previous studies provided evidence for an interaction between CD4⁺ and CD8⁺ T cells after cryo-thermal therapy, but the mechanism remains unclear, especially pertaining to how CD8⁺ T cells promote the Th1 differentiation of CD4⁺ T cells. This study revealed that activated CD4⁺ and CD8⁺ T cells are critical for CTT-induced antitumor immunity, and the interaction between activated T cells is enhanced. The reciprocal regulation of activated CD8⁺ and CD4⁺ T cells was through LFA-1/ICAM-1 interactions, in which CD8⁺ T cells facilitate Notch1-dependent CD4⁺ Th1-dominant differentiation and promote IL-2 secretion of CD4⁺ T cells. Meanwhile, IL-2 derived from CD4⁺ T cells enhances the cytotoxicity of CD8⁺ T cells and establishes a positive feedback loop via increasing the expression of LFA-1 and ICAM-1 on T cells. Clinical analyses further validated that LFA-1/ICAM interactions between CD4⁺ and CD8⁺ T cells are correlated with clinical outcomes. Our study extends the functions of the LFA-1/ICAM-1 adhesion pathway, indicating its novel role in the interaction of CD4⁺ and CD8⁺ T cells. Full article

Migraine is a chronic neurovascular disease with unclear pathophysiological mechanisms. In this study, its pathogenic mechanisms were investigated through bioinformatics analysis of migraine-related pathways and key genes. Female Sprague Dawley rats were divided into control and migraine model groups. The control group received saline, while the migraine model group received nitroglycerin (NTG) to induce migraines over four weeks. Migraine-like behaviors were assessed within two hours following the final NTG injection. Genes of hypothalamus were identified using DESeq2. Gene ontology enrichment and KEGG pathway analyses were conducted, followed by the identification of hub genes based on protein interaction networks by using algorithms such as Closeness, Degree, and Maximum Neighborhood Component. Rats with NTG-induced migraine showed increased head scratching and cage climbing and a reduced sucrose preference. Transcriptome analysis revealed 1564 differentially expressed genes, with 1233 upregulated and 331 downregulated. Pathways linked to inflammation, PI3K–Akt signaling, and cytokine–cytokine receptor interactions were found to have enriched expression of several genes. Further protein interaction network analysis identified nine hub genes: Alb, Tgfb1, Cd4, Ptprc, Itgb1, Icam1, Col1a1, Pxdn, and Itgad. These findings suggest that migraine involves PI3K–Akt signaling and cytokine–cytokine receptor interactions, providing insights into molecular mechanisms and potential therapeutic targets. However, the study was limited by a small sample size and reliance on a single experimental model, which may constrain the clinical applicability of the findings. Full article

Non-pathogenic natural and recombinant strains of human Enteroviruses are the subject of ongoing study with some strains having been approved for use as anticancer agents. The efficacy of oncolytic virotherapy depends upon identifying the receptor utilized by a specific strain for cell entry, and the presence of this receptor on the surface of cancer cells. Accordingly, a rapid and straightforward approach to determining the enteroviral receptors is necessary for developing an effective patient-specific, virus-based cancer therapy. To this end, we created a panel of seven lines with double knockouts on the background of the HEK293T cell line, which lacks the IFNAR1 gene. In these lines, the main viral receptor genes, including PVR, CXADR, CD55, ITGA2, SCARB2, ICAM1, and FCGRT, were knocked out using the CRISPR/Cas9 system. The panel of lines was validated on twelve different Enteroviruses types, providing a basis for studying the molecular mechanisms of enterovirus entry into cells, and for developing new therapeutic strains. Full article

The protease, a disintegrin and metalloproteinase with thrombospondin type 1 motif member 13 (ADAMTS13), known to cleave only the von Willebrand factor (VWF), has powerful regulatory effects on microvascular platelet adhesion, thrombosis, inflammation, and endothelial dysfunction. We study the protection against diabetes-induced retinal injury in experimental rats by supplementation with recombinant ADAMTS13. We compare human epiretinal membranes and vitreous samples from nondiabetic subjects and patients with proliferative diabetic retinopathy (PDR) and extend in vitro analyses with the use of various immunodetection and spectrofluorimetric methods on rat retina and human retinal glial and endothelial cell cultures. Functional studies include the assessment of the blood–retinal barrier (BRB), cell adhesion, and in vitro angiogenesis. In epiretinal membranes, endothelial cells and monocytes/macrophages express ADAMTS13. The levels of VWF, the platelet marker CD41, ADAMTS13, and the biomarkers of endothelial cell injury soluble VE-cadherin and soluble syndecan-1 are increased in PDR vitreous. ADAMTS13 is downregulated in diabetic rat retinas. The intravitreal administration of ADAMTS13 attenuates diabetes-induced BRB breakdown, the downregulation of VE-cadherin and β-catenin, and the upregulation of VWF, CD41, phospho-ERK1/2, HMGB1, VCAM-1, and ICAM-1. In Müller cells, ADAMTS13 attenuates MCP-1, MMP-9, and ROS upregulation induced by diabetic mimetic conditions. In HRMECs, ADAMTS13 attenuates the shedding of the soluble VE-cadherin and soluble syndecan-1 and the levels of phospho-ERK1/2, MCP-1, fractalkine, and ROS induced by diabetic mimetic conditions, the upregulation of ICAM-1 and VCAM-1 elicited by TNF-α, the adherence of monocytes induced by TNF-α, and VEGF-induced migration of human retinal microvascular endothelial cells. Our findings suggest that enhancing ADAMTS13 levels in situ ameliorates diabetes-induced retinal inflammation and vascular dysfunction. Full article

Objectives: Reduced expression of adhesion molecules in tumor vasculature can limit infiltration of effector T cells. To improve T cell adhesion to tumor endothelial cell (EC) antigens and enhance transendothelial migration, we developed bispecific, T-cell engaging antibodies (bsAb) that activate T cells after cross-linking with EC cell surface antigens. Methods: Recombinant T-cell stimulatory anti-VEGFR2–anti-CD3 and costimulatory anti-TIE2–anti-CD28 or anti-PD-L1–anti-CD28 bsAb were engineered and expressed. Primary lines of human umbilical vein endothelial cells (HUVEC) that constitutively express VEGFR2 and TIE2 growth factor receptors and PD-L1, but very low levels of adhesion molecules, served as models for anergic tumor EC. Results: In cocultures with HUVEC, anti-VEGFR2–anti-CD3 bsAb increased T cell binding and elicited rapid T cell activation. The release of proinflammatory cytokines TNF-α, IFN-γ, and IL-6 was greatly augmented by the addition of anti-TIE2–anti-CD28 or anti-PD-L1–anti-CD28 costimulatory bsAb. Concomitantly, T cell-released cytokines upregulated E-selectin, ICAM1, and VCAM1 adhesion molecules on HUVEC. HUVEC cultured in breast cancer cell-conditioned medium to mimic the influence of tumor-secreted factors were similarly activated by T cell-engaging bsAb. Migration of T cells in transwell assays was significantly increased by anti-VEGFR2–anti-CD3 bsAb. The combination with costimulatory anti-TIE2–anti-CD28 bsAb augmented activation and proliferation of migrated T cells and their cytotoxic capacity against spheroids of the MCF-7 breast cancer cell line seeded in the lower transwell chamber. Conclusions: T cells activated by anti-VEGFR2–anti-CD3 and costimulatory EC-targeting bsAb can reverse the energy of quiescent EC in vitro, resulting in improved T cell migration through an EC layer. Full article

Traumatic optic neuropathy (TON) has been regarded a vision-threatening condition caused by either ocular or blunt/penetrating head trauma, which is characterized by direct or indirect TON. Injury happens during sports, vehicle accidents and mainly in military war and combat exposure. Earlier, we have demonstrated that remote ischemic post-conditioning (RIC) therapy is protective in TON, and here we report that AMPKα1 activation is crucial. AMPKα1 is the catalytic subunit of the heterotrimeric enzyme AMPK, the master regulator of cellular energetics and metabolism. The α1 isoform predominates in immune cells including macrophages (Mφs). Myeloid-specific AMPKα1 KO mice were generated by crossing AMPKα1^Flox/Flox and LysM^cre to carry out the study. We induced TON in mice by using a controlled impact system. Mice (mixed sex) were randomized in six experimental groups for Sham (mock); Sham (RIC); AMPKα1^F/F (TON); AMPKα1^F/F (TON+RIC); AMPKα1^F/F LysM^Cre (TON); AMPKα1^F/F LysM^Cre (TON+RIC). RIC therapy was given every day (5–7 days following TON). Data were generated by using Western blotting (pAMPKα1, ICAM1, Brn3 and GAP43), immunofluorescence (pAMPKα1, cd11b, TMEM119 and ICAM1), flow cytometry (CD11b, F4/80, CD68, CD206, IL-10 and LY6G), ELISA (TNF-α and IL-10) and transmission electron microscopy (TEM, for demyelination and axonal degeneration), and retinal oxygenation was measured by a Unisense sensor system. First, we observed retinal morphology with funduscopic images and found TON has vascular inflammation. H&E staining data suggested that TON increased retinal inflammation and RIC attenuates retinal ganglion cell death. Immunofluorescence and Western blot data showed increased microglial activation and decreased retinal ganglion cell (RGCs) marker Brn3 and axonal regeneration marker GAP43 expression in the TON [AMPKα1^F/F] vs. Sham group, but TON+RIC [AMPKα1^F/F] attenuated the expression level of these markers. Interestingly, higher microglia activation was observed in the myeloid AMPKα1^F/F KO group following TON, and RIC therapy did not attenuate microglial expression. Flow cytometry, ELISA and retinal tissue oxygen data revealed that RIC therapy significantly reduced the pro-inflammatory signaling markers, increased anti-inflammatory macrophage polarization and improved oxygen level in the TON+RIC [AMPKα1^F/F] group; however, RIC therapy did not reduce inflammatory signaling activation in the myeloid AMPKα1 KO mice. The transmission electron microscopy (TEM) data of the optic nerve showed increased demyelination and axonal degeneration in the TON [AMPKα1^F/F] group, and RIC improved the myelination process in TON [AMPKα1^F/F], but RIC had no significant effect in the AMPKα1 KO mice. The myeloid AMPKα1c deletion attenuated RIC induced anti-inflammatory macrophage polarization, and that suggests a molecular link between RIC and immune activation. Overall, these data suggest that RIC therapy provided protection against inflammation and neurodegeneration via myeloid AMPKα1 activation, but the deletion of myeloid AMPKα1 is not protective in TON. Further investigation of RIC and AMPKα1 signaling is warranted in TON. Full article

Angiotensin II (Ang II) is an effective vasoconstriction peptide, a major effector molecule of the renin–angiotensin–aldosterone system (RAAS) and one of the important causes of endothelial dysfunction. Ferroptosis is considered to be involved in the occurrence and development of cardiovascular diseases. This study is dedicated to exploring the role and mechanism of Ang II-induced ferroptosis in HUVECs and to finding molecular targets for vascular endothelial injury and dysfunction during the progression of hypertension. In this study, we found that with the increase in exposure concentration, the intracellular ROS content and apoptosis rate increased significantly, the NO release decreased significantly in the medium- and high-concentration groups and the ET-1 content in the high-concentration group increased significantly. The expression of ZO-1 protein was significantly decreased in the high-concentration group. The expression of p-eNOS, VE-cadherin and Occludin protein showed a dose-dependent downward trend, while the ICAM-1 protein showed an upward trend. Ang II caused lipid metabolism disorders in HUVECs, and the PL–PUFAs associated with ferroptosis were significantly increased. In addition, Ang II promoted a significant increase in intracellular free Fe²⁺ content and MDA and a significant decrease in GSH content. Furthermore, the expression of GPX4, SLC7A11 and SLC3A2 was down-regulated, the expression of ACSL4, LPCAT3 and ALOX15 was up-regulated, and the ratio of p-cPLA2/cPLA2 was increased. After the intervention of ferroptosis inhibitor Fer-1, the injury and dysfunction of HUVECs induced by Ang II were significantly rescued. Immunofluorescence results showed that the expression of CD36 showed a significant increasing trend and was localized in the cytoplasm. Over-expression of CD36 promoted Ang II-induced ferroptosis and endothelial dysfunction. In conclusion, Ang II induces the injury of HUVECs, decreases vascular diastole and endothelial barrier-related molecules, and increases vascular constriction and adhesion-related molecules, which may be related to CD36 and its mediated lipid peroxidation and ferroptosis signals. Full article

Intercellular adhesion molecule 1 (ICAM-1/CD54), a transmembrane glycoprotein, has been considered as one of the most important adhesion molecules during leukocyte recruitment. It is encoded by the ICAM1 gene and plays a central role in inflammation. Its crucial role in many inflammatory diseases such as ulcerative colitis and rheumatoid arthritis are well established. Given that neuroinflammation, underscored by microglial activation, is a key element in neurodegenerative diseases such as Parkinson’s disease (PD), we investigated whether ICAM-1 has a role in this progressive neurological condition and, if so, to elucidate the underpinning mechanisms. Specifically, we were interested in the potential interaction between ICAM-1, glial cells, and ferroptosis, an iron-dependent form of cell death that has recently been implicated in PD. We conclude that there exist direct and indirect (via glial cells and T cells) influences of ICAM-1 on ferroptosis and that further elucidation of these interactions can suggest novel intervention for this devastating disease. Full article

Glaesserella parasuis (GPS) can cause severe systemic inflammation in pigs, resulting in huge economic losses to the pig industry. At present, no effective method is available for the prevention and control of GPS infection. Molecular breeding for disease resistance is imminent, but disease-resistance genes have not been identified. To study the mechanism of systemic acute inflammation caused by GPS, we established three in vitro infection models (3D4/21 cells, PK15 cells, and PAVEC cells) according to its infection path. There was no significant difference in apoptosis among the three kinds of cells after 12 h of continuous GPS stimulation, while inflammatory factors were significantly upregulated. Subsequent transcriptome analysis revealed 1969, 1207, and 3564 differentially expressed genes (DEGs) in 3D4/21 cells, PK15 cells, and PAVEC cells, respectively, after GPS infection. Many of the DEGs were predicted to be associated with inflammatory responses (C3, CD44, etc.); cell proliferation, growth and apoptosis; gene expression; and protein phosphorylation. Key signaling pathways, including S100 family signaling, bacteria and virus recognition, and pathogen-induced cytokine storm signaling, were enriched based on Ingenuity Pathway Analysis (IPA). Furthermore, a total of three putative transmembrane receptors and two putative G-protein-coupled receptors, namely F3, ICAM1, PLAUR, ACKR3, and GPRC5A, were identified by IPA among the three types of cells. ACKR3 and GPRC5A play pivotal roles in bacterial adhesion, invasion, host immune response and inflammatory response through the S100 family signaling pathway. Our findings provide new insights into the pathological mechanisms underlying systemic inflammation caused by GPS infection in pigs, and they lay a foundation for further research on disease-resistance breeding to GPS. Full article

Gastric cancer is the fourth leading cause of cancer deaths worldwide. The presence of chemoresistant cells has been used to explain this high mortality rate. These higher tumorigenic and chemoresistant cells involve cancer stem cells (CSCs), which have the potential for self-renewal, a cell differentiation capacity, and a greater tumorigenic capacity. Our research group identified gastric cancer stem cells (GCSCs) with the CD24+CD44+CD326+ICAM1+ immunophenotype isolated from gastric cancer patients. Interestingly, this GCSC immunophenotype was absent in cells isolated from healthy people, who presented a cell population with a CD24+CD44+CD326+ immunophenotype, lacking ICAM1. We aimed to explore the role of ICAM1 in these GCSCs; for this purpose, we isolated GCSCs from the AGS cell line and generated a GCSC line knockout for ICAM1 using CRISPR/iCas9, which we named GCSC-ICAM1^KO. To assess the role of ICAM1 in the GCSCs, we analyzed the migration, invasion, and chemoresistance capabilities of the GCSCs using in vitro assays and evaluated the migratory, invasive, and tumorigenic properties in a zebrafish model. The in vitro analysis showed that ICAM1 regulated STAT3 activation (pSTAT3-ser727) in the GCSCs, which could contribute to the ability of GCSCs to migrate, invade, and metastasize. Interestingly, we demonstrated that the GCSC-ICAM1^KO cells lost their capacity to migrate, invade, and metastasize, but they exhibited an increased resistance to a cisplatin treatment compared to their parental GCSCs; the GCSC-ICAM1^KO cells also exhibited an increased tumorigenic capability in vivo. Full article

The COVID-19 pandemic, which is caused by the SARS-CoV-2 virus, has resulted in extensive health challenges globally. While SARS-CoV-2 primarily targets the respiratory system, clinical studies have revealed that it could also affect multiple organs, including the heart, kidneys, liver, and brain, leading to severe complications. To unravel the intricate molecular interactions between the virus and host tissues, we performed an integrated transcriptomic analysis to investigate the effects of SARS-CoV-2 on various organs, with a particular focus on the relationship between renal failure and COVID-19. A comparative analysis showed that SARS-CoV-2 triggers a systemic immune response in the brain, heart, and kidney tissues, characterized by significant upregulation of cytokine and chemokine secretion, along with enhanced migration of lymphocytes and leukocytes. A weighted gene co-expression network analysis demonstrated that SARS-CoV-2 could also induce tissue-specific transcriptional profiling. More importantly, single-cell sequencing revealed that COVID-19 patients with renal failure exhibited lower metabolic activity in lung epithelial and B cells, with reduced ligand–receptor interactions, especially CD226 and ICAM, suggesting a compromised immune response. A trajectory analysis revealed that COVID-19 patients with renal failure exhibited less mature alveolar type 1 cells. Furthermore, these patients showed potential fibrosis in the hearts, liver, and lung increased extracellular matrix remodeling activities. However, there was no significant metabolic dysregulation in the liver of COVID-19 patients with renal failure. Candidate drugs prediction by Drug Signatures database and LINCS L1000 Antibody Perturbations Database underscored the importance of considering multi-organ effects in COVID-19 management and highlight potential therapeutic strategies, including targeting viral entry and replication, controlling tissue fibrosis, and alleviating inflammation. Full article