Search Results (123)

Background: T cell regeneration in the thymus is intrinsically linked to the T cell-biased lineage differentiation of hematopoietic stem and progenitor cells (HSPCs). Although nonhuman primates (NHPs) serve as indispensable models for studying thymic output under physiological and pathological conditions, a non-animal technology facilitating efficient TCR-selected T cell development and evaluating T cell output from NHP-derived HSPCs has been lacking. To address this gap, we established a rhesus macaque-specific artificial thymic organoid (RhATO) modeling primary thymus-tissue-free thymopoiesis. Methods: The RhATO was developed by expressing Rhesus macaque (RM) Delta-like Notch ligand 1 in mouse bone marrow stromal cell line (MS5-RhDLL1). The bone marrow-derived HSPCs were aggregated with MS5-RhDLL1 and cultured forming 3D artificial thymic organoids. These organoids were maintained under defined cytokine conditions to support complete T cell developmental ontogeny. T cell developmental progression was assessed by flow cytometry, and TCR-selected subsets were analyzed for phenotypic and functional properties. Results: RhATOs recapitulated the complete spectrum of thymopoietic events, including emergence of thymus-seeding progenitors, CD4⁺CD3⁻ immature single-positive and CD4⁺CD8⁺ double-positive early thymocytes, and mature CD4⁺ or CD8⁺ single-positive subsets. These subsets expressed CD38, consistent with the recent thymic emigrant phenotype, and closely mirrored canonical T cell ontogeny described in humans. RhATO-derived T cells were TCR-selected and demonstrated cytokine expression upon stimulation. Conclusions: This study provides the first demonstration of an NHP-specific artificial thymic technology that faithfully models thymopoiesis. RhATO represents a versatile ex vivo platform for studying T cell development, immunopathogenesis, and generating TCR selected T cells. Full article

Cancer-associated fibroblasts (CAFs) are key components of the tumor microenvironment (TME) that influence cancer progression via extracellular matrix (ECM) remodeling and secretion of growth factors and cytokines. Endothelial-to-mesenchymal transition (EndMT) is emerging as an important axis among the heterogeneous origins of CAFs. This review introduces the diverse methods used to induce EndMT in cancer—mouse tumor models, conditioned-medium treatment, co-culture, targeted gene perturbation, ligand stimulation, exosome exposure, irradiation, viral infection, and three-dimensional (3D) culture systems—and summarizes EndMT cell-type evidence uncovered using transcriptomic and proteomic technologies. Hallmark EndMT features include spindle-like morphology, increased motility, impaired angiogenesis and barrier function, decreased endothelial markers (CD31, VE-cadherin), and increased mesenchymal markers (α-SMA, FN1). Reported mechanisms include signaling via TGF-β, cytoskeletal/mechanical stress, reactive oxygen species, osteopontin, PAI-1, IL-1β, GSK-3β, HSP90α, Tie1, TNF-α, HSBP1, and NOTCH. Cancer-induced EndMT affects tumors and surrounding TME—promoting tumor growth and metastasis, expanding cancer stem cell-like cells, driving macrophage differentiation, and redistributing pericytes—and is closely associated with poor survival and therapy resistance. Finally, we indicate each study’s stance: some frame cancer-induced EndMT as a source of CAFs, whereas others, from an endothelial perspective, emphasize barrier weakening and promotion of metastasis. Full article

We identified Murashka, a RING finger protein, in an oogenesis screen as a regulator of Drosophila ovary germline stem cell niche development. Mutant alleles of murashka exhibited an enlarged niche phenotype reminiscent of increased Notch signalling and displayed genetic interactions with Notch alleles, and with polychaetoid, a regulator of Notch during niche development. These interactions uncovered both positive and negative impacts on Notch in different genetic backgrounds. In S2 cells, Murashka formed a complex with Notch and colocalised with Notch in the secretory pathway. Murashka expression in S2 cells down-regulated Notch signalling levels but could result in increased fold induction due to the proportionally greater decrease in basal ligand-independent activity. In vivo Murashka expression had different outcomes on different Notch target genes. We observed a decrease in the expression of vestigial along the anterior/posterior boundary of the wing imaginal disc, but not of wingless at the dorsal/ventral boundary. Instead, weak ectopic wingless was observed, which was synergistically increased by the coexpression of Deltex, a positive regulator of ligand-independent signalling. Our results identify a novel developmental role for murashka, a gene previously only associated with a function in long-term memory, and indicate a regulatory role for Murashka through a physical interaction with Notch that has context-dependent outcomes. Murashka adds to a growing number of ubiquitin ligase regulators which interact with Notch at different locations within its secretory and endocytic trafficking pathways. Full article

People engaged in various activities in cold environments—such as those living in cold climates, polar workers, cold storage workers, and athletes engaged in winter sports—are frequently affected by cold environments. Therefore, it is of great significance to explore the modelling and remodelling of bones in cold environments. Cold environments can shorten the length of bones, thin the thickness of bones, decrease bone mineral density (BMD), change the biomechanical properties of bones, and lead to bone loss. In addition, cold directly affects the bone microenvironment. Exposure to cold causes spindle-like and fibroblast-like changes in bone marrow mesenchymal stem cells (BMSCs) and decreases their proliferation, and cold exposure promotes the osteogenic differentiation of BMSCs partly through the p38 MAPK pathway. Cold also alters the dendritic differentiation of OBs by reducing the transmembrane glycoprotein E11/podoplanin and damages endothelial cells (ECs) by elevating levels of VEGF, resulting in a reduced blood supply and thus fewer OBs. In addition, cold promotes lipolysis of marrow adipose tissue (MAT), but in combination with exercise, it can promote the differentiation of BMSCs into MAT. Cold environments interfere with angiogenesis and inhibit bone growth by affecting factors such as platelet-derived growth factor type BB (PDGF-BB), slit guidance ligand 3 (SLIT3), Notch, and VEGF. In addition, cold environments may promote bone resorption by activating sympathetic nerves to activate β-adrenergic receptors and regulating leptin secretion, and regulate bone metabolism by activating the p38 MAPK signalling pathway and increasing the synthesis of brown fat, which ultimately inhibit bone formation and enhance bone resorption. In this paper, we describe the effects of cold environments on bones in the locomotor system in terms of bone structure, bone mass, biomechanical properties, and various skeletal cells, bone blood vessels, and bone fat systems in the bone microenvironment. Full article

Delta-like 1 homolog (DLK1), a non-canonical Notch ligand, is highly expressed in various malignant tumors, especially in hepatocellular carcinoma (HCC). CBA-1205 is an afucosylated humanized antibody against DLK1 with enhanced antibody-dependent cellular cytotoxicity (ADCC). The binding characteristics of CBA-1205 were analyzed by enzyme-linked immunosorbent assay and fluorescence-activated cell sorting assay. The ADCC activity of CBA-1205 was assessed. The anti-tumor efficacy of CBA-1205 was evaluated in xenograft mouse models, and toxicity and toxicokinetic profiles of CBA-1205 were evaluated in cynomolgus monkeys. CBA-1205 selectively bound to DLK1 among the Notch ligands and only to monkey and human DLK1. The binding epitope was between epidermal growth factor-like domains 1 and 2 of DLK1, which are not involved in any known physiological functions. The ADCC activity of CBA-1205 was confirmed using human peripheral blood mononuclear cells as effector cells. CBA-1205 as a single agent and in combination with lenvatinib demonstrated long-lasting anti-tumor efficacy, including tumor regression, in two liver cancer xenograft models. The toxicity and toxicokinetic profiles of CBA-1205 in cynomolgus monkeys were favorable. These findings suggest that CBA-1205 has the potential to be a useful therapeutic option for drug treatment in HCC. A phase 1 study is ongoing in patients with advanced cancers (jRCT2080225288, NCT06636435). Full article

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy with poor response to conventional therapy, derived from hematopoietic progenitors committed to T-cell lineage. Relapsed/Refractory patients account for nearly 20% of childhood and 45% of adult cases. Aberrant Notch signaling plays a critical role in T-ALL pathogenesis and therapy resistance. Notch inhibition is a promising therapeutic target for personalized medicine, and a variety of strategies to prevent Notch activation, including γ-secretase (GS) inhibitors (GSIs) and antibodies neutralizing Notch receptors or ligands, have been developed. Disruption of apoptosis is pivotal in cancer development and progression. Different reports evidenced the interplay between Notch and the anti-apoptotic Bcl-2 family proteins in T-ALL. Although based on early research data, this review discusses recent advances in directly targeting Notch receptors and the use of validated BH3 mimetics for the treatment of T-ALL and their combined action in light of current evidence of their use. Full article

The Notch signaling pathway plays a major role in embryological development and in the ongoing life processes of many animals. Its role is to provide cell-to-cell communication in which a Sender cell, bearing membrane-embedded ligands, instructs a Receiver cell, bearing membrane-embedded receptors, to adopt one of two available fates. Elucidating the evolution of this pathway is the topic of this paper, which uses an orthologs approach, providing a comprehensive basis for the study. Using BLAST searches, orthologs were identified for all the 49 components of the Notch signaling pathway. The historical time course of integration of these proteins, as the animals evolved, was elucidated. Insofar as cell-to-cell communication is of relevance only in multicellular animals, it is not surprising that the Notch system became functional only with the evolutionary appearance of Metazoa, the first multicellular animals. Porifera contributed a quarter of the Notch pathway proteins, the Cnidaria brought the total to one-half, but the system reached completion only when humans appeared. A literature search elucidated the roles of the Notch system’s components in modern descendants of the ortholog-contributing ancestors. A single protein, the protein tyrosine kinase (PTK) of the protozoan Ministeria vibrans, was identified as a possible pre-Metazoan ancestor of all three of the Notch pathway proteins, DLL, JAG, and NOTCH. A scenario for the evolution of the Notch signaling pathway is presented and described as the co-option of its components, clade by clade, in a repurposing of genes already present in ancestral unicellular organisms. Full article

Notch signaling, an important signaling pathway in cardiac development, has been shown to mediate the reparative functions of c-kit+ progenitor cells (CPCs). However, it is unclear how each of the four canonical Notch-activating ligands affects intracellular processes in c-kit+ cells when used as an external stimulus. Neonatal c-kit+ CPCs were stimulated using four different chimeric Notch-activating ligands tethered to Dynabeads, and the resulting changes were assessed using TaqMan gene expression arrays, with subsequent analysis by principal component analysis (PCA). Additionally, functional outcomes were measured using an endothelial cell tube formation assay and MSC migration assay to assess the paracrine capacity to stimulate new vessel formation and recruit other reparative cell types to the site of injury. Gene expression data showed that stimulation with Jagged-1 is associated with the greatest pro-angiogenic gene response, including the expression of VEGF and basement membrane proteins, while the other canonical ligands, Jagged-2, Dll-1, and Dll-4, are more associated with regulatory and epigenetic changes. The functional assay showed differential responses to the four ligands in terms of angiogenesis, while none of the ligands produced a robust change in migration. These data demonstrate how the four Notch-activating ligands differentially regulate CPC gene expression and function. Full article

Bone remodeling is an intricate process executed throughout one’s whole life via the cross-talk of several cellular events, progenitor cells and signaling pathways. It is an imperative mechanism for regaining bone loss, recovering damaged tissue and repairing fractures. To achieve this, molecular signaling pathways play a central role in regulating pathological and causal mechanisms in different diseases. Similarly, microRNAs (miRNAs) have shown promising results in disease management by mediating mRNA targeted gene expression and post-transcriptional gene function. However, the role and relevance of these miRNAs in signaling processes, which regulate the delicate balance between bone formation and bone resorption, are unclear. This review aims to summarize current knowledge of bone remodeling from two perspectives: firstly, we outline the modus operandi of five major molecular signaling pathways, i.e.,the receptor activator of nuclear factor kappa-B (RANK)-osteoprotegrin (OPG) and RANK ligand (RANK-OPG-RANKL), macrophage colony-stimulating factor (M-CSF), Wnt/β-catenin, Jagged/Notch and bone morphogenetic protein (BMP) pathways in regards to bone cell formation and function; and secondly, the miRNAs that participate in these pathways are introduced. Probing the miRNA-mediated regulation of these pathways may help in preparing the foundation for developing targeted strategies in bone remodeling, repair and regeneration. Full article

Microglia-mediated neuroinflammation has been linked to neurodegenerative disorders. Inflammation and aging contribute to microglial senescence. Microglial senescence promotes the development of neurodegenerative disorders, including Alzheimer’s disease (AD). In this study, we investigated the anti-neuroinflammatory and anti-senescence activity of Honokiol (HNK), a polyphenolic neolignane from Magnolia officinalis Rehder & E.H Wilson, in comparison with its synthetic analogue Honokiol Hexafluoro (CH). HNK reduced the pro-inflammatory cell morphology of LPS-stimulated BV2 microglia cells and increased the expression of the anti-inflammatory cytokine IL-10 with an efficacy comparable to CH. HNK and CH were also able to attenuate the alterations in cell morphology associated with cellular senescence in BV2 cells intermittently stimulated with LPS and significantly reduce the activity and expression of the senescence marker ß-galactosidase and the expression of p21 and pERK1/2. The treatments reduced the expression of senescence-associated secretory phenotype (SASP) factors IL-1ß and NF-kB, decreased ROS production, and abolished H2AX over phosphorylation (γ-H2AX) and acetylated H3 overexpression. Senescent microglia cells showed an increased expression of the Notch ligand Jagged1 that was reduced by HNK and CH with a comparable efficacy to the Notch inhibitor DAPT. Overall, our data illustrate a protective activity of HNK and CH on neuroinflammation and cellular senescence in microglia cells involving a Notch-signaling-mediated mechanism and suggesting a potential therapeutic contribution in aging-related neurodegenerative diseases. Full article

There is currently no effective treatment strategy for recurrent/metastatic adenoid cystic carcinoma (R/M ACC). Furthermore, recent single-agent and combination immunotherapy trials have failed in unselected ACC cohorts, unlike non-ACC salivary gland cancers. Genomic profiling revealed no actionable targets but NOTCH1 and KDM6A frameshift and CTCF splice site mutations (no MYB/L fusion) with a low tumor mutational burden (TMB), microsatellite stable (MSS) and negative programmed death ligand 1 (PD-L1) were observed. We recommended an anti-programmed cell death protein 1 (anti-PD-1) plus anti-Cytotoxic T-lymphocyte-associated protein 4 (anti-CTLA-4) combination based on TMB 2-fold greater-than-median TMB in ACC, tumor harboring multiple immunogenic frameshift or splice site mutations, and PD-L1 negativity. Accordingly, we achieved a complete response in a radiotherapy (RT) and chemotherapy (CT)-refractory patient with locally recurrent lacrimal gland (LG) ACC and lung metastasis following personalized immunotherapy in combination with integrative therapeutics. Therefore, it is crucial to assess not only conventional immune biomarkers but also patient-specific parameters, especially in “immune-cold” cancer types. Full article

Notch signaling is a conserved pathway crucial for nervous system development. Disruptions in this pathway are linked to neurodevelopmental disorders, neurodegenerative diseases, and brain tumors. Hairy/E(spl) (HES) genes, major downstream targets of Notch, are commonly used as markers for Notch activation. However, these genes can be activated, inhibited, or function independently of Notch signaling, and their response to Notch disruption varies across tissues and developmental stages. MIB1/Mib1 is an E3 ubiquitin ligase that enables Notch receptor activation by processing ligands like Delta and Serrate. We investigated Notch signaling disruption using the zebrafish Mib1 mutant line, mib1^ta52b, focusing on changes in the expression of Hairy/E(spl) (her) genes. Our findings reveal significant variability in her gene expression across different neural cell types, regions, and developmental stages following Notch disruption. This variability questions the reliability of Hairy/E(spl) genes as universal markers for Notch activation, as their response is highly context-dependent. This study highlights the complex and context-specific nature of Notch signaling regulation. It underscores the need for a nuanced approach when using Hairy/E(spl) genes as markers for Notch activity. Additionally, it provides new insights into Mib1’s role in Notch signaling, contributing to a better understanding of its involvement in Notch signaling-related disorders. Full article

Background. Alagille syndrome (ALGS) is a rare autosomal dominant genetic disease caused by pathogenic variants in two genes: Jagged Canonical Notch Ligand 1 (JAG1) and Notch Receptor 2 (NOTCH2). It is characterized by phenotypic variability and incomplete penetrance with multiorgan clinical signs. Methods. Using Next Generation Sequencing (NGS), we analyzed a panel of liver-disease-related genes in a population of 230 patients with cholestasis and hepatopathies. For the rare variants, bioinformatics predictions and pathogenicity classification were performed. Results. We identified eleven rare NOTCH2 variants in 10 patients, two variants being present in the same patient. Ten variants had never been described before in the literature. It was possible to classify only two null variants as pathogenic, whereas the most of variants were missense (8 out of 11) and were classified as uncertain significance variants (USVs). Among patients with ALGS suspicion, two carried null variants, two carried variants predicted to be pathogenic by bioinformatics, one carried a synonymous variant and variants in glycosylation-related genes, and two carried variants predicted as benign in the PEST domain. Conclusions. Our results increased the knowledge about NOTCH2 variants and the related phenotype, allowing us to improve the genetic diagnosis of ALGS. Full article

NOTCH3 receptor signaling has been linked to the regulation of smooth muscle cell proliferation and the maintenance of smooth muscle cells in an undifferentiated state. Pulmonary arterial hypertension (World Health Organization Group 1 idiopathic disease: PAH) is a fatal disease characterized clinically by elevated pulmonary vascular resistance caused by extensive vascular smooth muscle cell proliferation, perivascular inflammation, and asymmetric neointimal hyperplasia in precapillary pulmonary arteries. In this review, a detailed overview of the specific role of NOTCH3 signaling in PAH, including its mechanisms of activation by a select ligand, downstream signaling effectors, and physiologic effects within the pulmonary vascular tree, is provided. Animal models showing the importance of the NOTCH3 pathway in clinical PAH will be discussed. New drugs and biologics that inhibit NOTCH3 signaling and reverse this deadly disease are highlighted. Full article

Pulmonary arterial hypertension (PAH) is a progressive cardiopulmonary disease characterized by pathologic vascular remodeling of small pulmonary arteries. Endothelial dysfunction in advanced PAH is associated with proliferation, apoptosis resistance, and endothelial to mesenchymal transition (EndoMT) due to aberrant signaling. DLL4, a cell membrane associated NOTCH ligand, plays a pivotal role maintaining vascular integrity. Inhibition of DLL4 has been associated with the development of pulmonary hypertension, but the mechanism is incompletely understood. Here we report that BMPR2 silencing in pulmonary artery endothelial cells (PAECs) activated AKT and suppressed the expression of DLL4. Consistent with these in vitro findings, increased AKT activation and reduced DLL4 expression was found in the small pulmonary arteries of patients with PAH. Increased NOTCH1 activation through exogenous DLL4 blocked AKT activation, decreased proliferation and reversed EndoMT. Exogenous and overexpression of DLL4 induced BMPR2 and PPRE promoter activity, and BMPR2 and PPARG mRNA in idiopathic PAH (IPAH) ECs. PPARγ, a nuclear receptor associated with EC homeostasis, suppressed by BMPR2 loss was induced and activated by DLL4/NOTCH1 signaling in both BMPR2-silenced and IPAH ECs, reversing aberrant phenotypic changes, in part through AKT inhibition. Directly blocking AKT or restoring DLL4/NOTCH1/PPARγ signaling may be beneficial in preventing or reversing the pathologic vascular remodeling of PAH. Full article