Journal of Immunology Research

Alternaria fungi produce various structurally diverse mycotoxins, several of which exhibit immunomodulatory properties. Among these, alternariol monomethyl ether (AME), alternariol (AOH), alterperylenol (ALTP), altertoxin I (ATX-I), and altersetin (AST) have been reported to suppress lipopolysaccharide (LPS)-induced inflammatory responses. However, the precise molecular mechanisms underlying these effects remain unclear. The present study aimed to elucidate how these selected Alternaria mycotoxins (0.1-50 M) target the NF-{kappa}B signaling pathway in THP-1 monocytes. Key components of the NF-{kappa}B cascade were analyzed by immunofluorescence microscopy, Western blotting and qRT-PCR. Nuclear translocation of NF-{kappa}B p65 and its phosphorylated form (p- NF-{kappa}B p65) was assessed by Western blot, while cytokine responses were determined at transcript (qRT-PCR) and protein (ELISA) levels. Moreover, in silico docking analyses were performed to investigate potential interactions of the toxins with IKK{beta}, and receptor-mediated crosstalk was studied using the glucocorticoid receptor (GR) antagonist RU486. Co-treatment with RU486 attenuated the immunosuppressive effects of 1 and 5 M AOH, indicating partial involvement of GR-dependent mechanisms. AME, AOH, ALTP, ATX-I, and AST increased total I{kappa}B levels while reducing its phosphorylated form. Additionally, AST and ALTP decreased the protein levels of Toll-like receptor 4 (TLR4), the I{kappa}B kinase (IKK) complex, NF-{kappa}B p65, and p- NF-{kappa}B p65. While AOH (5 M) and AST (25 M) reduced nuclear translocation of p65 and p-p65, ALTP (2 M) enhanced nuclear localization despite decreasing cytokine expression. Together, these findings suggest toxin-specific interference at multiple regulatory levels of NF-{kappa}B signaling and provide novel mechanistic insight into the immunomodulatory effects of Alternaria mycotoxins.

Background: Natural essential oils exhibit antimicrobial and wound-healing properties, but their therapeutic application is limited by poor water solubility, volatility, and instability. This study developed and characterized a nanoemulsion of Ocimum gratissimum essential oil (OGNe) and evaluated its physicochemical properties, dermal safety, antibacterial activity, and wound-healing potential. Methods: Essential oil was obtained by hydrodistillation and formulated into nanoemulsions by high-speed stirring emulsification. Physicochemical properties, including pH, droplet size, polydispersity index, and storage stability, were determined. Acute dermal toxicity was assessed in Wistar rats following OECD Test Guideline 402. Antibacterial activity was evaluated using broth microdilution, minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC), and time-kill assays. Wound-healing efficacy was investigated using an excision wound model over 21 days using distilled water and trolamine serving as controls. Results: OGNe exhibited a stable milky appearance, near-neutral pH, and droplet sizes ranging from 26 to 224 nm. No signs of dermal toxicity or behavioral abnormalities were observed after topical administration. The nanoemulsion showed selective antibacterial activity, with the highest susceptibility against Acinetobacter baumannii (MIC = 1.125 L/mL), whereas Escherichia coli remained resistant. Time-kill assays demonstrated concentration-dependent bacteriostatic activity. In vivo, OGNe significantly accelerated wound contraction from day 3 onward (p < 0.0001), achieving healing rates comparable to or exceeding those of trolamine during the inflammatory and proliferative phases. Conclusion: Ocimum gratissimum nanoemulsions represent stable, biocompatible topical formulations that combine selective antibacterial activity with enhanced wound healing, supporting their potential as phytopharmaceutical nanoformulations for the management of acute skin wounds.

Influenza A viruses H3N2 and H10N5 represent, respectively, a persistently dominant seasonal pathogen and a newly documented zoonotic threat with the latter strain variants responsible for the first confirmed human fatality in January 2024, yet no vaccine platform currently addresses co-protection against both subtypes within a unified immunogen. We report here the immunoinformatics based vaccine design and multi-layered computational validation of a 419-amino-acid multi-epitope subunit vaccine construct targeting conserved hemagglutinin (HA) and neuraminidase (NA) antigens identified through multiple sequence alignment of the avian H10N5 (A/swine/Hubei/10/2008) and H3N2 human reference strain sequences to identify viral agents undergoing mammalian adaptations. Linear B-cell, cytotoxic T lymphocyte (CTL), and helper T lymphocyte (HTL) epitopes were predicted using ABCpred, BCEpred, BepiPred 2.0, NetMHCpan 2.1, and NetMHCpan 4.0, then filtered through VaxiJen 3.0, AllerTOP v2.1, and ToxinPred to retain only antigenic, non-allergenic, non-toxic candidates. The final construct, incorporating an avian {beta}-defensin N-terminal adjuvant with GPGPG, AAY, and EAAAK linkers, exhibited a molecular weight of 43.9 kDa, instability index of 31.15, and SOLPro solubility probability of 0.763. Tertiary structure modeling via I-TASSER and GalaxyRefine achieved 84.4% Ramachandran-favored residues. Molecular docking against TLR3 and TLR7 yielded binding free energies of -16.1 and -16.8 kcal/mol with picomolar dissociation constants. Molecular dynamics simulations confirmed complex stability over extended trajectories. Furthermore, codon optimization produced a Codon Adaptation Index of 1.0 for E. coli K12 expression. In silico immune simulation demonstrated robust activation of humoral and cellular immunity including elevated IgG1, IgM, IFN-{gamma}, IL-2, rapid NK cell expansion, and broad B-cell clonal diversity. These findings establish a computationally validated candidate capable of providing protection against influenza in multiple host organisms, warranting experimental advancement.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive malignancy characterized by a dense desmoplastic tumor microenvironment (TME) that limits drug penetration and promotes immune evasion. Effective treatment, therefore, requires simultaneous modulation of multiple signaling pathways. Here, we describe a directed polypharmacological strategy to identify dual modulators of c-MET and Smoothened (SMO), aiming to disrupt the protective stroma through SMO inhibition while directly suppressing tumor cell survival via c-MET targeting. An AI-guided virtual screening workflow combining the machine-learning platform PyRMD, trained on known c-MET and SMO ligands, with structure-based molecular docking was applied to a library of over 9 million compounds. This approach led to the identification of compound 21, an aminopyrimidine-benzamide-phenoxyquinoline derivative, as a dual c-MET/SMO inhibitor. Biochemical and cellular studies demonstrated that compound 21 selectively binds the SMO orthosteric site (pKi = 5.60), inhibits agonist-induced GLI (Glioma-associated oncogene) signaling (pIC50 = 5.50), and potently suppresses c-MET kinase activity (pIC50 = 6.94). Western blot analyses further revealed that compound 21 promotes ubiquitin-proteasome-mediated degradation of c-MET, eliminating receptor availability and limiting compensatory resistance signaling. In 3D heterotypic models comprising MIAPaCa2 pancreatic cancer cells and CAF154-hTERT fibroblasts, dual inhibition of SMO-mediated stromal support and c-MET-driven tumor progression resulted in greater cytotoxicity than the combination of the selective inhibitors Sonidegib and PHA-665752. Overall, compound 21 overcomes stromal-mediated resistance, enhances tumor cell death, and validates dual SMO/c-MET targeting as a promising single-agent therapeutic strategy for PDAC. One Sentence SummaryAn AI-identified dual SMO/c-MET inhibitor overcomes stromal resistance and degrades c-MET to suppress pancreatic cancer.

Glioblastoma remains a highly aggressive and therapy-resistant brain tumor, with limited benefit from the current standard-of-care regimen combining surgery, radiotherapy, and temozolomide. Overcoming chemoresistance therefore represents a critical unmet clinical need. Here, we investigate the anticancer potential of Succisa pratensis and its ability to enhance TMZ efficacy in GBM models. Treatment with S. pratensis markedly reduced cell proliferation and migration while significantly increasing sensitivity to TMZ. Integrated multi-omics analyses revealed extensive metabolic rewiring, characterized by suppression of central carbon metabolism and activation of stress-adaptive pathways. Mechanistically, we identify the Pregnane X Receptor, a key regulator of drug metabolism and chemoresistance, as a central node affected by treatment. Although S. pratensis increased PXR expression, this was not accompanied by induction of canonical downstream targets, including MDR1 and ALDH1A1, indicating a functional impairment of PXR transcriptional activity. Consistently, pharmacological inhibition of PXR using the antagonist SPA70 further potentiated the cytotoxic effects of S. pratensis and TMZ. Docking analyses suggest that specific secondary metabolites, including apigenin-derived compounds, may interact with the PXR ligand-binding domain, providing a potential molecular basis for this effect. Collectively, our findings indicate that S. pratensis enhances TMZ efficacy by inducing metabolic vulnerability and functionally impairing PXR signaling. These results highlight the therapeutic potential of plant-derived metabolites as adjuvant strategies to overcome chemoresistance in glioblastoma. Article HighlightsO_LISuccisa pratensis enhances temozolomide efficacy in glioblastoma by reducing proliferation, migration, and clonogenic growth. C_LIO_LIIntegrated proteomic and metabolomic analyses reveal extensive metabolic rewiring, with suppression of central carbon metabolism and induction of stress-adaptive pathways. C_LIO_LIPregnane X Receptor (PXR), a key regulator of chemoresistance, is functionally impaired despite increased expression, resulting in reduced activation of drug-resistance genes. C_LIO_LIPharmacological inhibition of PXR further potentiates the antitumor effects of Succisa pratensis and temozolomide, promoting apoptotic cell death. C_LIO_LIApigenin-derived metabolites show high affinity for the PXR ligand-binding domain and emerge as promising candidates to overcome temozolomide resistance in glioblastoma. C_LI

Androgen receptor (AR) antagonists, such as enzalutamide, suppress prostate cancer (PCa) cells to achieve temporary therapeutic effects. In addition to tumor cell-autonomous suppressive function, AR antagonists can also potentially exert anti-tumor effects via mitigating cytotoxic T cells exhaustion. However, strategies for effectively harnessing enzalutamides immunotherapeutic effects remain elusive. In studying a recently described glutamine antagonist prodrug (DRP-104) in PCa models, we found that despite the initial response, tumors ultimately became resistant. Intriguingly, compared to the untreated (DRP-104 treatment-naive) tumors, the resistant tumors became highly susceptible to enzalutamide in vivo. Additionally, treating tumors with DRP-104 and enzalutamide simultaneously also yielded superior therapeutic efficacy. We demonstrated that DRP-104 therapy promoted the infiltration of CD8+ T cells as well as regulatory T cells (Treg) in responsive tumors, and the tumor-infiltrating Treg were mostly depleted upon enzalutamide treatments. Mechanistically, we showed that Treg differentiation from mouse CD4+ T cells was attenuated by enzalutamide. We further demonstrated that Treg induction was accompanied by the interaction between AR and aryl hydrocarbon receptor (AhR), the nuclear receptor indispensable for Treg differentiation, in the nuclei of CD4+ T cells, and this interaction was diminished by enzalutamide. In further support of AR signaling in Treg biogenesis, analysis of available gene expression datasets found that AR expression was elevated in Treg when compared to CD4+ T cells in human peripheral blood mononuclear cells (PBMCs). In addition, it was positively correlated with Treg module scores in several human cancer types. Finally, using an anti-GPC3 (Glypican 3) vaccination model, we demonstrated that CD4+ T cells subjected to Treg induction in the presence of enzalutamide were less effective in protecting GPC3-expressing tumor cells from CD8+ T cells cytotoxic killing. Collectively, these results suggest that AR promotes Treg s differentiation and/or immunosuppressive functions, and nominate enzalutamide as a Treg-mitigating agent for potentiating immunotherapies. Our results also demonstrate that an otherwise unintended, Treg-promoting property of DRP-104 can be leveraged to unleash the immune-regulatory function of enzalutamide for the treatment of PCa.

Background & Aims: Hepatocellular carcinoma (HCC) remains a leading cause of cancer mortality, with most patients presenting at advanced stages requiring systemic therapy. Despite promising outcomes with immune checkpoint inhibitors (ICI), responses remain variable due to an immunosuppressive tumor microenvironment. Y90 radioembolization offers potential immune priming, but only a subset of patients benefit. Here, we apply spatial multi-omics to delineate baseline and treatment-induced immune features and identify predictive signatures of progressive disease (PD) for early detection of patients unlikely to benefit from therapy. Approach & Results: Paired baseline (Day 0) and on-treatment (Day 35) biopsies were obtained from 33 patients, following Y90 radioembolization (Day 14) and nivolumab. Multiplex immunohistochemistry (mIHC) was used for cell-cell interaction analysis. A subset was further profiled using Visium (n=13) for tissue category-specific analysis and NanoString GeoMx DSP (n=12) for cell type-resolved transcriptomic and pathway analyses. Global spatial transcriptomics analysis revealed minimal baseline immune activity in PD, indicating an intrinsically immune-deficient TME. Despite treatment-induced activation, PD exhibited reduced CD8+ T cell abundance and limited reinvigoration of exhausted subsets, and persistent LAG-3-associated exhaustion. DSP showed downregulation of antigen presentation and T cell activation pathways. Macrophage profiling revealed enrichment of CD38+ phenotypes, contrasting CXCL9-CXCR3-associated responses in responders. Furthermore, a 72-gene PD signature was identified and validated in TCGA, associating with poorer survival. Conclusions: Integrated spatial multi-omics reveals that PD in HCC is associated with an immune-deficient TME, characterized by LAG-3-associated CD8+ exhaustion and immunosuppressive macrophages. A 72-gene signature enables early identification and supports alternative therapeutic strategies.

Blood group antigens, defined by epitopes on the erythrocyte surface, are central to transfusion safety and maternal-fetal compatibility. While the genetic basis of many clinically relevant blood group antigens is well established, which structural and biophysical parameters determine whether a single-nucleotide variant gives rise to an antigenic phenotype remains unclear. Here, we integrate structural, biophysical, and evolutionary analyses to systematically evaluate features associated with single amino acid substitutions across 24 human protein-based blood group systems. We analyse 319 variants with curated phenotypic annotations alongside 481 control variants, identifying key determinants of null and antigenic phenotypes. Null variants are characterized by high evolutionary conservation, burial within the protein core, loss of hydrophobicity, increased polarity, and a propensity for arginine substitutions. Antigenic variants are also enriched in arginine; however, in contrast to null variants, they tend to occur at less conserved, more solvent-accessible, and structurally flexible sites. Supervised machine learning models trained on structural and biophysical descriptors were applied to distinguish (i) null and (ii) antigenic variants from controls, achieving balanced accuracies of 0.82 and 0.63, respectively. Feature importance analysis identified predicted pathogenicity, solvent accessibility, and evolutionary conservation as the most predictive determinants of null variants, whereas hydrophobicity, conservation, and flexibility dominated antigen prediction. This work establishes a framework linking molecular variation to blood group phenotypes and provides a foundation for predicting the impact of novel missense mutations in transfusion medicine and beyond.

Tenuazonic acid (TeA) is an emerging Alternaria mycotoxin frequently detected in food and feed commodities, raising concerns about its toxicological relevance. Chronic oral exposure to TeA has been reported to induce dysplastic alterations in the esophageal mucosa of mice, while human biomonitoring data indicate an association between TeA exposure and esophageal cancer, although a causal relationship has not yet been established. At a mechanistic level, the effects of TeA in esophageal cells remain poorly characterized. Therefore, this study investigated the impact of TeA on cytotoxicity, oxidative stress, DNA damage, mitochondrial homeostasis, cell-cycle distribution and transcriptomic stress responses in human esophageal KYSE-510 cells. TeA induced a concentration-dependent reduction in metabolic activity and total protein content after 24 h exposure to 0.1-100 M. Significant cytotoxicity was measured starting from 20 M. At sub-cytotoxic concentrations, TeA triggered rapid ROS formation within 5-30 min exposure and induced formamidopyrimidine-DNA glycosylase (FPG) sensitive DNA damage after 1 h exposure (5-7.5 M), indicating oxidative DNA lesions. In addition, TeA altered mitochondrial morphology after 4 h exposure at 7.5 M, manifested by shrinkage of the mitochondrial network area and perinuclear redistribution, while mitochondrial respiration showed only a non-significant tendency towards reduced respiratory capacity. RNA sequencing after 6 h exposure to 10 M TeA revealed oxidative stress-associated transcriptional changes, impaired antioxidant and stress-adaptive responses, and p53-associated stress signaling. Furthermore, TeA induced significant G2/M phase accumulation after 24 h exposure to 1-10 M.

Mycobacterium tuberculosis (Mtb) infection kills more people worldwide than any other pathogen. While the Bacille Calmette-Guerin (BCG) vaccine for Mtb has been widely used for over a century, it provides insufficient protection to eradicate this disease. One of our labs has recently established that a protein antigen (H1) can be combined with a STING pathway agonist to achieve strong protection against Mtb in mice, with performance that exceeds that of the BCG vaccine. However, its reliance on a synthetic cyclic dinucleotide (CDN) with relatively poor cell uptake requires higher dosing levels, thus increasing costs. To increase the efficiency of this vaccine and provide a delivery strategy that could also be used in humans, the H1 Mtb antigen and CDN adjuvant were conjugated to genome-free MS2 viral capsids that included cationic mutations to increase cell uptake. Specifically, the H1 antigen was conjugated to the external surface of MS2 using a tyrosinase-mediated oxidative coupling reaction, and the native STING agonist cGAMP was coupled to internal cysteine residues through a reductively cleavable disulfide linker. The resulting MS2-H1 and MS2-cGAMP conjugates were then co-delivered for three doses of vaccination in mice before exposure to Mtb. The MS2-based vaccine platform was observed to have comparable efficacy to the original H1/CDN formulation, but its enhanced uptake properties enabled 57-fold less CDN and 3-fold less H1 antigen. Additionally, this vaccine elicited immune responses that have been previously demonstrated to correlate with protection. The ability of the capsid shells to protect the CDN cargo during transport allowed enzymatically produced, and thus readily accessible, cGAMP to be used instead of more costly CDNs that require many synthetic steps. This, combined with the reduced overall amount of CDN and H1 that was required, could lower the production costs of future vaccines substantially. Finally, the ability of the capsid-based carriers to bypass the membrane transporters for CDNs suggests that this enhanced vaccination platform is likely to exhibit improved human efficacy in future studies.

Invasive pulmonary aspergillosis poses a life-threatening complication in immunocompromised individuals, including recipients of allogeneic hematopoietic cell transplantation (allo-HCT). By contrast, immunocompetent individuals are usually protected against infection with Aspergillus fumigatus, the causative agent of aspergillosis. The mechanisms underlying pulmonary innate immune protection remain poorly understood. Here, we identify alveolar macrophages (AMs) as key players in pulmonary antifungal defense. In immunocompromised mice, AMs conferred protection against lethal invasive aspergillosis by day 6, but not day 4 post-allo-HCT. To enhance AM function at the earlier time point, we tested cytokine-based interventions and showed that M-CSF, but not IL-34, which both bind to the CSF-1 receptor, promotes migratory activity, phagolysosomal function and fungal killing in both mouse and human primary tissue-resident AMs. In allo-HCT recipient mice, M-CSF treatment preserved lung tissue integrity, suppressed pro-inflammatory cytokines, and protected mice from lethal invasive aspergillosis. The M-CSF-driven protective effect was abrogated upon AM depletion. Our findings demonstrate a critical role of tissue-resident AMs in pulmonary antifungal immunity and suggest that therapeutic modulation of AM activity via M-CSF may offer a promising strategy to combat severe fungal infections in immunocompromised patients.

Background: Diabetic kidney disease (DKD) remains a leading cause of end-stage renal disease worldwide, characterized by progressive structural and metabolic alterations secondary to chronic hyperglycemia. While numerous type 1 and type 2 rodent models have been developed to study the pathophysiology of DKD, no single model perfectly recapitulates the full clinical spectrum of human disease. The selection of an optimal model depends deeply on the specific research objective, as phenotypic expression and histopathological severity vary significantly across different strains and induction methods. The present study provides a comparative analysis of the renal histological of three widely utilized murine models: the chemically induced streptozotocin (STZ) model and the genetic Akita (type 1) and db/db (type 2) models. Methods: Male STZ-induced (28 weeks post-induction), heterozygous Akita (28 weeks old), and db/db mice at two different age intervals (18-21 and 16-24 weeks old) were assessed. Renal injury was quantified using four light-microscopic parameters: glomerulomegaly, mesangial hypercellularity, tubular vacuolization and arteriolar hyalinosis. Due to observed discrepancies between metabolic and structural findings in the db/db strain, transmission electron microscopy (TEM) was employed for subcellular characterization. Results: All models exhibited significant hyperglycemia and albuminuria. At the light-microscopic level, STZ and Akita mice demonstrated consistent and pronounced renal lesions. In contrast, db/db mice despite increasing albuminuria and obesity, light microscopy revealed heterogeneous and inconsistent histopathological changes. However, TEM analysis of db/db mice kidneys successfully captured early ultrastructural injury, including irregular glomerular basement membrane (GBM) thickening and focal podocyte foot process effacement, which were undetectable by light microscopy. Conclusions: Our findings indicate that the Akita and STZ-induced models exhibit prominent structural alterations detectable by conventional light microscopy, whereas the db/db model requires ultrastructural evaluation by TEM to reliably confirm renal injury. This study underscores the limitation of routine histology in certain type 2 diabetes models and highlights the complementary value of TEM for accurate histopathological characterization. Collectively, the alternative histopathological markers identified herein offer sensitive and readily accessible indices for monitoring early-to-moderate DKD progression, providing a more robust framework for preclinical model selection and therapeutic evaluation in future studies.

Introduction. TAC/SARIFA has been introduced as a new robust and easy-to-evaluate biomarker in several cancer entities, including colorectal cancer. It is defined by direct contact between at least five tumour cells and one adipocyte and is believed to indicate metabolic reprogramming associated with adverse outcome. However, the mechanism that leads to TAC/SARIFA positivity remains unclear. To investigate whether there is an individual component, we conducted a study on double and triple cancers, establishing a within patient design. Methods. We retrospectively analysed a total of 135 cases with 276 colorectal cancers from two academic medical centres. The TAC/SARIFA status was evaluated, as were the basic histopathological factors. The median follow-up time was 120 months. Results. Cases with any TAC/SARIFA positive tumours showed significantly reduced overall survival (62 vs. 88 months; p = 0.011). Analysing the entire cohort, the rates of concordant and discordant cases followed a random distribution. However, restricting the analysis to synchronous pT3/4 cases revealed a significant deviation from a random distribution (p = 0.016). Conclusion. This study reveals significant concordance of TAC/SARIFA status in synchronous locally advanced colorectal double/triple carcinomas, supporting the concept that tumour adipocyte interaction reflects a host related microenvironmental condition linked to metabolic reprogramming rather than a purely tumour intrinsic event.

Objective We developed a patient- and joint-specific explant co-culture system to model active psoriatic arthritis (PsA) and capture donor-specific tissue responses to therapeutic interventions. Methods Based on convergent joint pathology between end-stage osteoarthritis (OA) and PsA, OA cartilage-bone and synovium tissues from arthroplasty patients were exposed to synovial fluid (SF) obtained from PsA and OA patients. Histological outcomes (synovitis, proteoglycan distribution), curated gene expression, soluble mediators, and proteinase activity were assessed over 7-21-days. Model responses to dexamethasone (DEX) and the anti-tumor necrosis factor antibody adalimumab (ADA) were evaluated. Results PsA SF induced distinct inflammatory and tissue remodeling responses compared to OA SF and control conditions, including altered cartilage proteoglycan distribution, increased synovitis, and tissue-specific transcriptional changes. Multivariate analyses identified distinct osteochondral and synovial transcriptional responses to PsA SF, characterized by reduced osteochondral COL2A1 expression and increased synovial expression of inflammatory and matrix-remodeling genes, including MMP1 and CXCL8. DEX and ADA elicited donor-specific responses across histological, transcriptional, and protein readouts. Among multivariable model outputs, histologic synovitis scores emerged as the most clinically aligned parameter, demonstrating associations with baseline PsA donor disease activity, active joint counts, pain, high-sensitivity C-reactive protein (hsCRP), and radiographic scores. Synovitis score changes to DEX and ADA treatments also aligned with corresponding PsA SF donor clinical improvements to corticosteroid and TNF-modifying therapies. Conclusion This osteochondral-synovial explant co-culture model captured donor-specific inflammatory and treatment-responsive features of PsA SF-induced pathology, thereby providing a clinically relevant ex vivo platform for studying patient-specific therapeutic responses in PsA.

Pancreatic cancer remains a highly lethal malignancy with limited therapeutic options. Chimeric antigen receptor (CAR) therapy has revolutionized the treatment of hematological cancers but still faces major limitations in solid tumors, particularly due to the scarcity of tumor-specific targets. Cutaneous lymphocyte antigen (CLA) recently emerged as a promising PDAC target due to its high tumor expression and limited presence in healthy tissues. However, previously reported CLA-directed CAR constructs lacked antitumor functionality. Here, we investigated multiple strategies to generate functional CLA-targeting CAR approaches. We first hypothesized that impaired activity resulted from fratricide caused by CLA expression on activated T cells. CLA knockout was successfully achieved through deletion of fucosyltransferase-7, but not by knockout of the major CLA carrier backbones CD162, CD44 or CD43, suggesting additional CLA carriers or compensatory regulation. As CLA knockout alone did not restore CAR-mediated killing, we explored whether insufficient binding affinity limited CAR activity. Affinity maturation was performed in silico and in vitro using yeast surface display, identifying 39 candidate mutations, although none restored cytotoxicity. We finally switched to an AdCAR strategy using anti-biotin CAR T cells combined with biotinylated anti-CLA scFv-Fc adapters. This approach enabled efficient, concentration-dependent cytotoxicity with both CLA-targeting binders. Additionally, we identified a dynamic, cell density-dependent regulation of CLA expression. Finally, glycan profiling of CLA binders further revealed broader-than-expected glycan interactions, suggesting a potentially wider definition of the CLA family. Overall, our findings establish CLA as a functional PDAC immunotherapy target while revealing unexpected complexity in its regulation and molecular presentation.

BACKGROUND: Perfluorooctane sulfonate (PFOS) is a widely distributed persistent organic pollutant in the environment and has been associated with an increased risk of atherosclerosis. However, the underlying pathogenic mechanisms remain largely unclear. This study aimed to investigate the effects of PFOS on atherosclerosis and its associated gut-vascular axis. METHODS: Pseudo-germ-free mouse models and fecal microbiota transplantation (FMT) were used to determine the role of the gut microbiota in PFOS-induced atherosclerosis. Metagenomic sequencing was performed to characterize alterations in gut microbial composition following PFOS exposure, and targeted metabolomics was used to assess bile acid profiles in the ileum and plasma. Transcriptomic analysis of Bacteroides caecimuris (B.caecimuris) was conducted to explore the reasons for the increased abundance of B.caecimuris after PFOS exposure. In addition, intestinal transcriptomics and ChIP-qPCR were performed to validate transcriptional regulation within the FXR-TLR3 signaling axis. RESULTS: Among 127 participants with paired serum and fecal samples, including 82 patients undergoing coronary angiography with Gensini scores (GS score), fecal PFOS levels were significantly associated with lipid profiles and GS score, whereas serum PFOS showed no such association. Mechanistically, PFOS exposure promotes intestinal enrichment of B. caecimuris by upregulating its tolC gene, thereby enhancing efflux capacity. This microbial shift was accompanied by reduced levels of tauro-ursodeoxycholic acid (TUDCA) and aberrant activation of intestinal FXR signaling. Further analyses demonstrated that FXR activation upregulated TLR3 expression and promoted inflammatory responses and atherosclerosis progression via the TLR3-NF-{kappa}B signaling axis. Both intestinal epithelial-specific FXR deficiency (Fxr{Delta}IE) and TUDCA supplementation significantly suppressed pathway activation and alleviated disease phenotypes.Functional experiments identified TLR3 as a key downstream effector of FXR. Overexpression of TLR3 abolished the protective effects observed in Fxr{Delta}IE mice. Moreover, pharmacological inhibition of TLR3 using CU CPT-4a significantly improved established atherosclerotic lesions in vivo. CONCLUSIONS: This study identifies a gut microbiota-driven FXR-TLR3 signaling axis that mediates PFOS-induced atherosclerosis. These findings provide new mechanistic insights into environmentally induced cardiovascular disease and suggest potential targets for risk assessment and therapeutic intervention.

Background: Abdominal aortic aneurysms (AAA) are characterized by dilation of the aorta that can lead to aortic rupture and death. The transcriptional co-activators Yes-Associated Protein (YAP) and WW-domain-containing transcriptional co-activator with PDZ-binding motif (TAZ) are mechanosensitive effectors of the highly conserved Hippo signaling pathway. It is hypothesized that cell-specific YAP/TAZ signaling in endothelial cells (EC) plays a pivotal role in mediating AAA formation and rupture. Methods: Single-cell RNA-sequencing in human AAAs was performed and differentially expressed genes (DEGs) were identified in the endothelial cell cluster. YAP/TAZ mRNA and protein expression were also assessed in human AAA and control aortic tissue. Two established murine AAA models were used with male C57BL/6 and EC-CreERT2-YAPfl/fl/TAZfl/fl mice with/without Verteporfin (VPF, YAP/TAZ inhibitor) and XMU-MP-1 (YAP/TAZ activator) treatments. On postoperative days 14 and 28, aortic diameter, histology, cytokine, and MMP2 expressions were evaluated. Results: A significant alteration in EC-specific differentially expressed YAP/TAZ-related genes was observed in which 242 genes were upregulated and 71 genes were downregulated in AAA compared to controls. Human AAA tissue showed a significant increase in YAP and TAZ protein expressions compared to controls. Elastase-treated EC-YAP/TAZ-/- mice showed a significant decrease in AAA diameter compared to littermate controls. Histological quantification revealed preservation of -smooth muscle actin, reduced elastin fiber breaks, and decreased macrophage infiltration in EC-YAP/TAZ-/- mice compared to littermate controls. Importantly, pharmacological inhibition of YAP/TAZ using VPF significantly attenuated AAAs in two experimental murine models. In vitro data demonstrates that VPF inhibits endothelial cell YAP expression, downregulating pathways associated with pathogenic angiogenesis and vascular inflammation. Conclusions: These data suggest that EC-specific YAP/TAZ signaling mediates AAA formation. Pharmacological inhibition of the Hippo pathway can significantly mitigate aortic inflammation and vascular remodeling to decrease the progression of AAAs and prevent aortic rupture.

Blockade by sedimentary particles, such as mineral crystals, is a continuous risk the kidney tubule faces. To prevent that, kidney resident macrophages form transepithelial protrusions and remove intratubular sedimentary particles, a behavior particularly prevailing in the medulla over the cortex. However, the molecular mechanisms underlying this characteristic behavior of medulla macrophages are incompletely understood. In this study, we identified that the medulla had higher mechanical stiffness than the cortex in steady state, which was further elevated when kidney stone formed. Increased tissue rigidity was sensed by medulla macrophages via mechanoreceptor Piezo1, which promoted macrophage protrusion formation and their ability to clean the tubules. Loss of Piezo1 expression in kidney macrophages predisposed mice to intratubular accumulation of mineral crystal in steady state and accelerated kidney stone formation during oxalate intake challenge. Signaling via Piezo1 mobilized molecules involved in cell adhesion and protrusion assembly, including Talin2 and focal adhesion kinase (FAK). Finally, we developed a first-of-its-kind cell-based therapy for the treatment of experimental nephrolithiasis by exploiting macrophage Piezo1 activity, and this strategy shows great promise for future translational research.

Extrahepatic delivery of lipid nanoparticles (LNPs) to non-phagocytic cells is a major challenge, with the leading strategy involving surface functionalization with target-specific monoclonal antibody (mAb) ligands. We investigate the stability of mAb-conjugated LNPs using two anchoring systems: the commonly used DSPE-PEG2kDa-maleimide and a block copolymer, PCL5kDa-b-PEG2kDa -maleimide, with the hypothesis that conjugation to a 150,000 Da antibody could overwhelm the relatively small ~600 Da aliphatic anchor on the PEG-lipid in vivo. Shedding of the mAB would compromise targeting. Conjugation integrity following IV injection was assessed by tagging LNPs and mAbs with metal ion tracers that could be quantified by ICP-MS. Results show that DSPE-PEG-mAb rapidly (within 1h) dissociates from LNPs in blood, leading to accelerated LNP clearance. In contrast, mAbs conjugated using PCL-b-PEG remained stably associated with the LNP over the 24h circulation and clearance of the construct. Results are connected to a thermodynamic model that reproduces experimental findings for PEG-anchor(-mAb) shedding in vitro and in vivo. This study identifies anchoring strength as a critical, unconsidered parameter for in vivo performance when conjugating mAbs to LNPs for extrahepatic delivery.

The incidence of early-onset colorectal cancer (CRC) has risen sharply in recent decades1, yet the biological basis underlying the distinct behavior of tumors arising in young versus aged tissues remains poorly understood. Here we show that aging reprograms the epigenetic landscape of the colon, restricting colon tumor growth through stable silencing of developmental and fetal gene programs. We find that colon tumors arising in aged mice are intrinsically less proliferative than those arising in young animals. Multi-omic profiling of normal colon and colon tumors reveals that aging drives DNA hypermethylation, loss of Polycomb-associated chromatin states, and reduced chromatin accessibility at a defined set of developmental genes that are bivalent (marked by both H3K27me3 and H3K4 methylation), transcriptionally active in colon tumors from young animals and repressed in both tumors and normal tissue from old animals. Among the genes most strongly repressed in old animals is Tacstd2 (Trop2), a regulator of fetal intestinal programs and epithelial stemness. Pharmacologic inhibition of DNA methylation reactivates the aging-silenced gene network in organoids from old animals, whereas genetic disruption of Tacstd2 suppresses growth and developmental transcriptional programs in young tumor organoids. TACSTD2, fetal gene signatures, and the aging-associated bivalent gene program are likewise repressed in late-onset vs. early-onset human colorectal cancers. Collectively, these findings identify age-associated epigenetic silencing of developmental gene programs as a causal mechanism that constrains colorectal tumor growth and provide a mechanistic framework for understanding the distinct biology of early-onset colorectal cancer.