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The recently identified proton-activated chloride (PAC) channel is ubiquitously expressed, and it regulates several proton-sensitive physiological and pathophysiological processes. While the PAC channel is activated by strong acids due to the binding of protons to extracellular binding sites, here, we describe the way in which weak acids inhibit the PAC channel by a mechanism involving a distinct extracellular binding site. Whole-cell patch clamp was performed on wildtype HEK293T cells, PAC-knockout HEK293 cells expressing human (h)PAC mutant constructs, and on hiPSC-derived cardiomyocytes. Proton-induced cytotoxicity was examined in HEK293T cells. Acetic acid inhibited endogenous PAC channels in HEK 293T cells in a reversible, concentration-dependent, and pH-dependent manner. The inhibition of PAC channels was also induced by lactic acid, propionic acid, itaconic acid, and β-hydroxybutyrate. Weak acids also inhibited recombinant wildtype hPAC channels and PAC-like currents in hiPSC-derived cardiomyocytes. Replacement of the extracellular arginine 93 by an alanine (hPAC–Arg93Ala) strongly reduced the inhibition by some weak acids, including arachidonic acid. Although lactic acid inhibited PAC, it did not reduce the proton-induced cytotoxicity examined in wildtype HEK 293 cells. To conclude, weak acids inhibit PAC via an extracellular mechanism involving Arg93. These data warrant further investigations into the regulation of the PAC channel by endogenous weak acids.

Arteriovenous malformations (AVMs) are congenital vascular anomalies defined by abnormal direct connections between arteries and veins due to their complex structure or endovascular approaches. Pharmacological strategies targeting the underlying molecular mechanisms are thus gaining increasing attention in an effort to determine the mechanism involved in AVM regulation. In this study, we examined 30 human tissue samples, comprising 10 vascular samples, 10 human fibroblasts derived from AVM tissue, and 10 vascular samples derived from healthy individuals. The pharmacological agents thalidomide, U0126, and rapamycin were applied to the isolated endothelial cells (ECs). The pharmacological treatments reduced the proliferation of AVM ECs and downregulated miR-135b-5p, a biomarker associated with AVMs. The expression levels of angiogenesis-related genes, including VEGF , ANG2 , FSTL1 , and MARCKS , decreased; in comparison, CSPG4 , a gene related to capillary networks, was upregulated. Following analysis of these findings, skin samples from 10 AVM patients were reprogrammed into induced pluripotent stem cells (iPSCs) to generate AVM blood vessel organoids. Treatment of these AVM blood vessel organoids with thalidomide, U0126, and rapamycin resulted in a reduction in the expression of the EC markers CD31 and α-SMA. The establishment of AVM blood vessel organoids offers a physiologically relevant in vitro model for disease characterization and drug screening. The authors of future studies should aim to refine this model using advanced techniques, such as microfluidic systems, to more efficiently replicate AVMs’ pathology and support the development of personalized therapies.

Due to the complexity of modeling tumor-host interactions within the tumor microenvironment in vitro, we developed a 3D heterotypic cellular breast cancer (BC) model. We generated spheroid models using MCF7, MDA-MB-231, and SK-BR-3 cell lines alongside cancer-associated (BrC4f) and normal (BN120f) fibroblasts in ultra-low attachment plates. Stromal spheroids (3Df) were formed using a liquid overlay technique (graphical abstract). The YT cell line and peripheral blood NK (PB-NK) cells were used as immune components in our 3D model. In this study, we showed that stromal cells promoted tumor cell aggregation into spheroids, regardless of the initial proliferation rates, with NK cells accumulating in fibroblast-rich regions. The presence of CAFs within the model induced alterations in the expression levels of MICA/B and PD-L1 by tumor cells within the 3D-2 model. The feasibility of utilizing a 3D cell BC model in combination with cytokines and PB-NKs was evaluated. We observed that IL-15 and IL-2 enhanced NK cell activity within spheroids, whereas TGFβ had varying effects on proliferation depending on the cell type. Stimulation with IL-2 and IL-15 or TGFβ1 altered PB-NK markers and stimulated their differentiation into ILC1-like cells in 3D models. These findings underscore the regulatory function of CAFs in shaping the response of the tumor microenvironment to immunotherapeutic interventions.

HexaBody-CD27 (GEN1053/BNT313) is an investigational novel agonistic CD27 antibody engineered to enhance T-cell costimulation and promote antitumor immunity. Through the introduction of a hexamerization-enhancing mutation in the IgG Fc domain, HexaBody-CD27 was designed to drive clustering and activation of CD27 via intermolecular Fc:Fc interactions between membrane-bound antibodies, independent of crosslinking by FcγR-bearing cells. HexaBody-CD27 carries an Fc-silencing mutation to prevent T-cell depletion through Fc-mediated effector functions. In vitro, HexaBody-CD27 induced CD27 receptor signaling independent of FcγR-mediated crosslinking in a reporter assay. It also enhanced T-cell proliferation, cytotoxic activity and proinflammatory cytokine secretion in primary human lymphocytes. In contrast to benchmark IgG1 CD27 antibodies, HexaBody-CD27 did not induce phagocytosis of T cells in vitro. HexaBody-CD27 promoted ex vivo tumor infiltrating lymphocyte (TIL) expansion in non-small cell lung cancer (NSCLC) specimens, in particular of CD8 + TILs. The combination of HexaBody-CD27 with an anti-PD-1 antibody enhanced T-cell proliferation, cytokine secretion, and cytotoxic activity in vitro compared to either compound alone. In conclusion, HexaBody-CD27 enhanced T-cell activation and effector functions in an FcγR-crosslinking-independent manner, without inducing T-cell depletion. The immune agonist activity of HexaBody-CD27 was potentiated in combination with PD-1 blockade.

Peroxisome proliferator-activated receptor gamma (PPARG) is a nuclear receptor family transcription factor (TF) critical for adipogenesis, lipid metabolism, insulin sensitivity, and inflammation. It has also been known to play essential roles in trophoblast development and placentation. Dysregulation of PPARG in trophoblast differentiation has been implicated in pregnancy complications, such as pre-eclampsia and gestational diabetes. However, the molecular mechanisms of PPARG-dependent target gene regulation and its interactions with other regulatory factors during human trophoblast differentiation remain unclear. Using human trophoblast stem cells (TSCs), mimicking placental cytotrophoblasts (CTs), and their differentiation into extravillous trophoblasts (EVTs) as our models, we reveal that PPARG has cell-type-specific targets in TSCs and EVTs. We also find that while PPARG is essential for both TSC self-renewal and EVT differentiation, only its role in EVT differentiation is ligand sensitive and requires ligand-binding domain (LBD)-mediated transcriptional activity, whereas its function in TSC self-renewal appears to be ligand insensitive. Combined analysis with chromosomal targets of previously defined key TFs in TSCs and EVTs shows that PPARG forms trophoblast cell-type-specific regulatory circuitries, leading to differential target gene regulation via transcriptional re-wiring during EVT differentiation. Additionally, the enhanced invasiveness of EVTs treated with a PPARG agonist suggests a potential connection between PPARG pathways and human placenta accreta.

The hypoxic tumor microenvironment, particularly hypoxia-conditioned cancer-associated fibroblasts (CAFs), drives breast cancer (BC) progression and therapy resistance. However, the molecular mechanisms linking hypoxic CAFs to BC plasticity and chemoresistance remain elusive. Primary CAFs were isolated from high-grade BC tissues (Grade III) and characterized (α-SMA⁺/CD34⁻/pan-CK⁻), with normal fibroblasts (NFs) from reduction mammoplasty as controls. Hypoxic CAF-derived exosomal circSTAT3 stability was validated using RNase R resistance and actinomycin D assays. Exosomes were characterized via transmission electron microscopy (TEM), dynamic light scattering (DLS), and marker profiling (CD63⁺/TSG101⁺/Alix⁺, calnexin⁻). Functional effects of hypoxic CAF exosomes on TNBC cells (MDA-MB-231, SUM159) were assessed through proliferation/migration assays, stemness/epithelial-mesenchymal transition (EMT) marker analysis, and siRNA-mediated circSTAT3 knockdown. Mechanistic studies employed luciferase assays and RNA immunoprecipitation (RIP). Chemoresistance was evaluated by cisplatin half-maximal inhibitory concentration (IC₅₀). In vivo tumor growth and stemness enrichment were analyzed in xenografts. Clinical validation used BC tissues (n = 60) and plasma exosomes from BC patients (n = 40) versus healthy controls (n = 25). Hypoxic CAF-derived exosomes efficiently transferred circSTAT3 to TNBC cells, promoting proliferation, migration, EMT, and stemness marker expression. SiRNA-mediated circSTAT3 knockdown reversed these effects. Mechanistically, circSTAT3 acted as a competitive endogenous RNA (ceRNA), sponging miR-671-5p to derepress NOTCH1. Hypoxic CAF exosomes increased cisplatin IC₅₀ in TNBC cells, while circSTAT3 depletion restored chemosensitivity. In vivo, hypoxic CAF exosomes accelerated tumor growth, enriched CD44⁺/NOTCH1⁺ populations, and elevated circulating exosomal circSTAT3. Clinically, circSTAT3 was significantly upregulated in advanced BC tissues (p < 0.01) and patient plasma exosomes (p < 0.01), correlating with lymph node metastasis. This study identifies a hypoxia-driven feedforward loop wherein CAF-derived exosomal circSTAT3 promotes TNBC stemness and chemoresistance via miR-671-5p/NOTCH1 signaling. CircSTAT3 redefines stromal-tumor crosstalk as a circRNA-driven process and serves as both a circulating non-invasive biomarker and a promising therapeutic target to disrupt stromal-mediated resistance in aggressive TNBC. The online version contains supplementary material available at 10.1186/s12967-025-06794-8.

Background: Human cytomegalovirus (CMV) is a major pathogen that poses significant risks to immunocompromised individuals and neonates. The tegument protein pp71, encoded by the UL82 gene, plays a pivotal role in initiating viral lytic replication and evading host immune responses. Despite its clinical relevance, standardized monoclonal antibodies (mAbs) for pp71 remain limited, prompting the need to expand the available repertoire of antibodies targeting this critical protein. Methods: In this study, we constructed a diverse human single-chain variable fragment (scFv) library using RNA derived from the B cells of four healthy donors. The library was expressed in Saccharomyces cerevisiae , and iterative rounds of magnetic-activated cell sorting (MACS) were performed against recombinant pp71. Clonal enrichment was monitored using flow cytometry. Results: Among the isolated clones, one designated ID2 exhibited high sensitivity and specificity for pp71, as demonstrated by flow cytometry, immunofluorescence, an enzyme-linked immunosorbent assay (ELISA), and biolayer interferometry (BLI). Conclusions: Collectively, these findings establish a novel pp71-specific mAb and underscore the utility of yeast surface display combined with MACS for expanding the antibody toolkit available for CMV research and diagnostics.

Respiratory syncytial virus (RSV) infection in the upper respiratory tract promotes disease progression and transmission, with excessive inflammation contributing to severe lower respiratory tract involvement. This study investigates the immunomodulatory effects of Lactobacillus rhamnosus D3189 on viral kinetics and innate immune responses in well-differentiated nasal epithelial cells (WD-NECs). WD-NECs from healthy adult donors (N = 8) were cultured in vitro , treated with L. rhamnosus D3189, and then infected with RSV (strain RS4) 24 hours later. Viral replication and shedding were assessed via RT-qPCR and plaque assays. Cytotoxicity and epithelial integrity were evaluated using LDH release and transepithelial electrical resistance (TEER). Inflammatory and antiviral responses were investigated using multiplex immunoassays, AlphaLISA, and ELISA. RSV infection induced robust viral replication and shedding, disrupted epithelial barrier integrity, and triggered the release of pro-inflammatory cytokines and type I/III interferons. L. rhamnosus D3189 alone did not induce cytotoxicity or inflammation. While it had no effect on viral replication, TEER, LDH release, or IFN-λ1/3 levels, D3189 significantly enhanced IFN-β production, reduced viral shedding, and attenuated RSV-induced cytokine and chemokine responses. L. rhamnosus D3189 modulates the epithelial immune response to RSV, reducing inflammation and viral shedding without compromising epithelial integrity. These findings support its potential as a novel strategy to limit RSV-associated infection and transmission.