Search Results (300)

Background/Objectives: Therapeutic antibodies have become an important modality for cancer treatment; however, effective targeted antibody therapies for T-cell acute lymphoblastic leukemia (T-ALL) remain limited. CD99 is a promising therapeutic target due to its high expression in leukemic T cells and its capacity to transmit apoptotic signals. However, the efficacy of antibody-mediated apoptosis may depend on the CD99 region recognized by the antibody. This study compared the in vitro apoptotic activity of two chimeric anti-CD99 antibodies, ChAbMT99/1 and ChAbMT99/3, which recognize distinct regions of CD99 in T-ALL models. Methods: ChAbMT99/1 and ChAbMT99/3 were generated as human IgG1 antibodies. Antibody reactivity and binding site specificity were characterized using peptide-based ELISA. Binding to native CD99, apoptosis induction in two-dimensional (2D) Jurkat E6.1 and MOLT-4 suspension cultures and three-dimensional (3D) Jurkat E6.1-derived spheroid models, and cytotoxic effects on peripheral blood mononuclear cells (PBMCs) were assessed by flow cytometry. In vitro hematologic effects were assessed using hemagglutination and platelet aggregation assays. Results: ChAbMT99/1 and ChAbMT99/3 specifically recognized peptides corresponding to distinct CD99 regions, with peptide-binding EC₅₀ values of 0.813 and 0.819 μg/mL, respectively. Both antibodies exhibited binding reactivity to native CD99 on the tested cells. Functionally, both antibodies induced Annexin V/7-AAD-defined cell death in both 2D and 3D T-ALL models compared with controls in a crosslinking-dependent manner. In contrast, both antibodies induced low levels of cell death (<10%) in bulk PBMCs, with no visible hemagglutination or platelet aggregation observed in samples from five selected donors under the stated assay conditions. Conclusions: Both chimeric anti-CD99 antibodies demonstrated in vitro apoptotic activity against T-ALL models despite recognizing distinct regions of CD99. These findings provide a rationale for further investigation of their other mechanisms of action to support the future development of CD99-targeted antibody therapy for T-ALL. Full article

Objective: Self-assembling peptide nanomaterials are increasingly investigated as modular platforms for vaccine delivery and immune modulation. However, their structure-dependent interactions with immune cells remain insufficiently defined, limiting their early evaluation as peptide nanomaterial. This study evaluated the physicochemical properties and immunological effects of three Sup35 G7-derived self-assembling peptide nanomaterials, G7, G7d, and G7d(PEG)HER2, with particular attention to innate immune compatibility and macrophage responses. Methods: The morphology and surface charge of the peptides were characterized by transmission electron microscopy and zeta-potential analysis. Their cytocompatibility was assessed in THP-1 macrophages, Jurkat T cells, and IM-9 B cells. Transcriptomic and proteomic analyses were integrated to identify immune-cell-specific molecular responses. Cellular uptake, apoptosis, cytokine secretion, and intracellular reactive oxygen species production were further examined in macrophages. In vivo immunization in BALB/c mice was conducted to assess systemic immunoglobulin responses. Results: G7 and G7d assembled into large crystalline aggregates with limited dispersibility, whereas PEGylated G7d(PEG)HER2 formed more uniform nanofibers. All three materials showed positive zeta potentials and relatively low cytotoxicity across the tested immune cell models. Among these cells, macrophages displayed the strongest molecular responses. Integrated transcriptomic and proteomic analyses revealed coordinated alterations mainly involving metabolic remodeling, redox homeostasis, proteasome activity, and mitochondrial-associated pathways, rather than predominant activation of classical pro-inflammatory signaling. Ultrastructural analysis confirmed intracellular uptake of assembled peptides and mitochondrial morphological changes without detectable apoptosis. Cytokine profiling showed reduced secretion of multiple cytokines and chemokines, while G7 induced higher intracellular reactive oxygen species than G7d and G7d(PEG)HER2. In vivo, G7d administration was associated with increased serum IgG levels, whereas G7 showed a moderate effect and G7d(PEG)HER2 exhibited minimal changes; IgM levels remained unchanged. Conclusions: Sup35 G7 based self-assembling peptides exhibit low acute toxicity but induce structure-dependent modulation of macrophage function. Differences in assembly morphology and surface modification may influence systemic humoral responses. These findings support the importance of physicochemical design in evaluating immune compatibility of peptide nanomaterial scaffolds. Full article

Ankylosing spondylitis (AS) is a chronic inflammatory disease strongly associated with human leukocyte antigen B27 (HLA-B27). However, the incomplete penetrance of HLA-B27 suggests that additional molecular mechanisms contribute to AS pathogenesis. Circular RNAs (circRNAs) have emerged as important regulators of immune responses, but their roles in AS pathogenesis remain incompletely understood. In this study, circRNA expression profiles were examined by high-throughput RNA sequencing and selected circRNAs were validated by quantitative RT-PCR in T cells from AS patients and healthy controls. Among the validated circRNAs, circRNPS1 was significantly up-regulated in T cells from AS patients. Overexpression of circRNPS1 in activated Jurkat cells increased IL-2, IL-17A, and interferon-γ expression and enhanced JAK2/STAT1 and MEK1/2-ERK signaling. RNA pull-down followed by proteomic analysis identified heterogeneous nuclear ribonucleoprotein K (hnRNPK) that binds to the back-splicing junction region of circRNPS1. CircRNPS1 increased the stability of hnRNPK and VAV1, whereas hnRNPK knockdown attenuated circRNPS1-mediated MEK1/2-ERK activation and inflammatory cytokine expression. Furthermore, an antisense RNA fragment targeting the back-splicing junction region of circRNPS1 suppressed IL-2, IL-17A, and interferon-γ expression in activated circRNPS1-overexpressing Jurkat cells and T cells from AS patients. Disrupting the interaction between circRNPS1 and hnRNPK therefore warrants further investigation as a potential therapeutic strategy for AS. Full article

Chemotherapy-induced immune dysregulation remains a major challenge in patients with advanced solid tumors. β-Glucan has been reported to possess immunomodulatory properties; however, its longitudinal effects on immune homeostasis during chemotherapy remain unclear. In this prospective, randomized phase II study, patients receiving chemotherapy were assigned to receive β-glucan powder (with L-glutamine and bioactive protein from bovine colostrum), β-glucan capsules (with L-glutamine), or standard care. Clinical outcomes, serial hematologic parameters, longitudinal immune profiling, and an in vitro Jurkat T-cell viability assay were evaluated. Compared with controls, β-glucan supplementation improved the disease control rate, accelerated neutrophil recovery, and maintained a more stable neutrophil-to-lymphocyte ratio during chemotherapy. Longitudinal immune profiling demonstrated greater stability of T-cell, NK-cell, NKT-cell, and KIR (CD158)-expressing immune-cell populations in β-glucan supplementation groups, suggesting attenuation of chemotherapy-induced immune remodeling. Both β-glucan formulations showed comparable immunomodulatory effects, although the expression of CD158b(+) and CD158i(+) NK-cell subsets remained more stable in the powder group during later treatment. In vitro, β-glucan significantly reduced Jurkat T-cell viability, indicating direct biological activity in addition to its clinical immunomodulatory effects. These findings suggest that β-glucan supplementation helps preserve immune homeostasis during chemotherapy and may serve as a promising adjunctive immunonutritional strategy for patients with advanced solid tumors. Full article

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy that requires novel therapeutic targets and selective repurposed drugs with low systemic toxicity. Here, we evaluated the clinically approved M3 muscarinic receptor antagonist, Darifenacin (DF), against Jurkat and MOLT-4 T-ALL cells, as well as healthy human T lymphocytes, under identical conditions. DF induced selective, concentration-dependent cytotoxicity with IC₅₀ values of 26.7 ± 1.07 µM (Jurkat) and 30.5 ± 1.54 µM (MOLT-4), whereas healthy T lymphocytes exhibited an apparent IC₅₀ of 197.9 ± 1.29 µM, corresponding to a 6.5–7.4-fold therapeutic window. Mechanistically, DF decreased M3 receptor and choline acetyltransferase expression and increased acetylcholinesterase activity. Convergent multi-assay validation confirmed that this cytotoxicity was driven by activation of the intrinsic mitochondrial apoptotic pathway, as evidenced by increased Bax, decreased Bcl-2, and cleavage of the executioner caspase-3. Furthermore, DF induced glycative stress through the accumulation of methylglyoxal (MG) and advanced glycation end products (AGEs), and selectively inhibited lysosomal Cathepsins B and C. Molecular docking predicted highly favorable molecular binding within the catalytic cavities of these proteases. These findings suggest that DF exhibits selective antileukemic activity by simultaneously disrupting cholinergic signaling, inducing glycative stress, inhibiting cathepsins, and triggering apoptosis. Thus, DF emerges as a promising candidate for multi-target drug repurposing in T-ALL therapy. Full article

Staphylococcus aureus is a major bacterial pathogen that can cause clinical infections and foodborne illnesses through the production of 25 exotoxin types. The most frequently implicated toxins in food poisoning outbreaks are Staphylococcal enterotoxins type A–E (SEA-SEE), which are the first enterotoxins discovered. While in vitro detection methods are available to identify the presence of enterotoxins, they cannot distinguish between biologically active and inactive forms of the toxins. Detection of biologically active enterotoxins currently relies on in vivo testing, using the emetic response in kittens or monkeys. Here, we show the development of an in vitro assay to detect the active forms of SEB, a potential biological warfare agent and leading cause of food poisoning, and SEC1, a frequent cause of staphylococcal food poisoning. The novel assay involves the implementation of a genetically engineered Jurkat T-cell line expressing TCR Vβ3, resulting in a dose response of IL-2 production when exposed to active toxin. We also show that at a concentration of 100 ng/mL, the biological activity of SEB is significantly decreased at temperatures over 70 °C, while pasteurization at 63 °C only slightly reduces the biological activity of the toxin. Our studies provide an alternative method to animal testing to determine the presence of active toxins and provide possible inactivation methods of the toxins. Full article

With the widespread coexistence of multiple food additives, their combined exposure has raised increasing health concerns. This study used high-content imaging to investigate the individual and combined effects of five common food additives on lipid metabolism in HepG2, Caco-2, and Jurkat T cells. In HepG2 and Caco-2 cells, all additives dose-dependently induced lipid and free cholesterol accumulation; by contrast, Jurkat T cells exhibited only sporadic statistical differences without biologically relevant dose-dependent effects. Notably, lipid droplet accumulation appeared as a relatively early and readily detectable response, though its suitability as a definitive biomarker of metabolic disturbance warrants further validation. Combined-exposure experiments, conducted in HepG2 and Caco-2 cells, revealed cell-specific interaction patterns: additive interactions dominated in HepG2 cells, while sodium benzoate and sodium cyclamate showed synergistic disruption in Caco-2 cells. These results provide in vitro evidence that multi-component combined exposure causes cell-specific lipid-related perturbations not fully captured by single-additive assessment, suggesting that mixture toxicity models should be incorporated to improve food safety evaluation. Full article

A systematic optimization of throughput and operational run time in Sorting by Interfacial Tension (SIFT) is presented. Reducing droplet size and enabling a broader distribution of droplet trajectories increased the number of droplets processed per sorting element, resulting in about a four-fold improvement in throughput from 30 to 125 droplets per second. Throughput was further enhanced through device parallelization, as demonstrated by devices incorporating two and four independent sorting regions. These configurations distribute droplets evenly across sorting elements. The elements exhibited comparable pH sorting thresholds, indicating similar flow conditions and drag forces within each region. Among the designs evaluated, the two-element configuration provided the optimal balance of throughput, specificity, robustness, and simplicity. It achieved maximum throughputs of about 250 droplets per second. In many biological applications, only 1 in 20–30 droplets are occupied to minimize multiple-cell occupancy, resulting in an effective sorting rate of approximately 8 cells per second. Throughput and pH sorting thresholds were preserved for two hours of continuous cell sorting. The improved platform was applied to examine the relationship between cellular glycolysis and iron homeostasis at the single-cell level in activated Jurkat cells. It revealed a subpopulation of highly glycolytic cells with significantly elevated iron uptake, consistent with prior reports linking iron regulation and T cell metabolism. Collectively, these advances expand the scale, stability, and biological applicability of SIFT. These advances facilitate large-scale functional studies and the capture of rare, metabolically distinct, cell populations. Full article

Porous hydrogels are critical for tissue engineering and regenerative medicine, as they mimic the native extracellular matrix to support cell infiltration and mass transport. A common strategy for engineering pore structures involves the incorporation and subsequent removal of sacrificial porogen templates (e.g., crystals or microspheres). Although this approach offers excellent control over pore architecture, it often suffers from complex procedures and biosafety concerns arising from incomplete template removal. In this work, we present a simple, biocompatible, and versatile templating approach. By systematically investigating the coacervation parameters, we produced gelatin microspheres (GSs) with tunable diameters from 7 µm to 300 µm via a green, instrument-free, and scalable process. Using GSs of 20–160 µm as porogens, we obtained alginate hydrogels with adjustable viscoelasticity, stiffness, and pore sizes. We then validated two cell-loading strategies for bulk porous alginate hydrogels using immortalized human T (Jurkat) cells: (i) post-seeding into pre-formed pores supported high-density, long-term, and organized cell aggregates with >90% viability; (ii) in situ encapsulation (prior to pore formation) yielded >80% viability and preserved the cluster-forming growth characteristics of Jurkat cells. Moreover, composites of smaller GSs (7–20 µm) with alginate could be syringe-extruded into stable, sub-millimeter porous filaments, demonstrating the potential for 3D printing. Collectively, this work provides a promising platform for three-dimensional culture of immune cells. Full article

Background: T-cell acute lymphoblastic leukemia (T-ALL) remains a challenging malignancy with limited targeted therapies. Natural phenanthrene derivatives represent a promising source of antileukemic agents. Objective: We screened a library of natural phenanthrene-type compounds to identify cytotoxic leads in Jurkat T-ALL cells and investigated the mechanisms underlying their activity, including potential synergy with the proteasome inhibitor bortezomib (BTZ). Methods: Jurkat cells were treated with thirteen natural compounds at 10 and 20 µM for 48 h; cell viability was assessed by WST-1 cell viability assay. Dose–response curves were generated to calculate IC₅₀ values. Apoptosis was evaluated by Hoechst 33342/PI staining and Annexin V/PI flow cytometry. Synergy with BTZ was analyzed using a fixed-ratio combination index (CI) approach and IC₅₀ shift analysis. ER stress signaling was characterized by Western blotting, quantitative RT-PCR of UPR genes (GRP78, ATF6), and immunoprecipitation of GRP78 followed by ubiquitin immunoblotting. Results: Among the compounds screened, Cypripedin showed the most potent cytotoxicity with an IC₅₀ of 6.52 µM. It induced a dose-dependent increase in apoptosis. Combination with BTZ yielded a CI < 0.5 and reduced BTZ IC₅₀ from 3.43 to 1.88 ng/mL. Cypripedin activated the unfolded protein response (UPR), modulated key ER stress markers including GRP78, p-PERK, p-eIF2α, p-JNK, and ATF6, downregulated UPR gene transcripts, and promoted GRP78 ubiquitination. Molecular docking predicted strong binding of Cypripedin to the GRP78 ATPase domain (Vina score −7.630 kcal/mol), supporting its mechanism of action. Conclusion: Cypripedin induces apoptosis in Jurkat T-ALL cells, synergizes with BTZ, and modulates ER stress through GRP78 ubiquitination. These findings support its further development as a potential T-ALL therapeutic. Full article

Efferocytosis—the tightly regulated clearance of apoptotic cells by phagocytes—maintains tissue homeostasis and is impaired in chronic obstructive pulmonary disease (COPD), where it contributes to persistent inflammation and increases the risk of comorbidities, including lung cancer. Inhaled corticosteroids (ICS) and long-acting β2 agonists (LABAs) are cornerstones of COPD therapy, but their effects on efferocytosis and on the COPD–lung cancer interface are incompletely understood. The primary objective of this study was to determine whether the ICS fluticasone propionate and the LABA salmeterol xinafoate, alone or in combination at clinically informed concentrations (10⁻⁸–10⁻⁶ M; 10⁻⁴ M reserved for cytotoxicity screening), modulate efferocytic capacity and inflammatory cell survival across diverse phagocyte models. We performed standardized in vitro efferocytosis assays using murine peritoneal and alveolar macrophages, the murine macrophage line J774A.1, PMA-differentiated human THP-1 macrophages, human blood-derived neutrophils, and the human alveolar adenocarcinoma cell line A549. Apoptosis was induced in Jurkat T cells by UV irradiation (100 mJ/cm²) and in murine thymocytes by dexamethasone (1 µM, 4 h); apoptotic and necrotic populations were characterized by annexin-V/propidium iodide and Sytox Green/Hoechst H-33342 staining. Peritoneal macrophages showed the highest efferocytic activity (~75%), followed by J774A.1 (~75% at 24 h), THP-1 (~30% at 2 h; ~60% at 24 h), alveolar macrophages (~40%), and A549 cells (<20%). Neither fluticasone nor salmeterol, individually or in combination, significantly altered efferocytic capacity in any phagocyte tested (all ANOVA p > 0.26). Fluticasone (10⁻⁸ and 10⁻⁶ M) significantly improved 24 h neutrophil survival and reduced early apoptosis (p < 0.05) but did not translate this survival benefit into enhanced efferocytosis. Salmeterol was cytotoxic at 10⁻⁴ M and inactive at 10⁻⁸–10⁻⁶ M. These findings indicate that the established anti-inflammatory benefits of ICS/LABA in COPD do not extend to augmentation of efferocytosis in this acute, serum-free in vitro setting and that pharmacological restoration of efferocytosis in COPD—a defect implicated in the pathogenesis and progression of comorbid lung cancer—will likely require strategies targeting the efferocytic machinery itself (e.g., MerTK, Rac-1, MFG-E8) rather than relying on current inhaled therapy. Full article

Background: Cytokine-induced JAK–STAT signaling becomes dysregulated in chronic human diseases, including cancer and autoimmunity, and contributes to immune cell dysfunction. A cytokine-independent approach to activating STAT proteins could “hardwire” pro-survival and effector programs in immune cells to sustain function within diseased tissues. Engineered variants of the herpesvirus saimiri tyrosine kinase interacting protein (TIP) can recruit the SRC family kinase (SFK) LCK to drive STAT phosphorylation and activation. Here, we evaluated the interactome of a TIP-derived, cytokine-independent STAT5 activator and determined whether it could induce STAT5 activation in immune cell lines and primary human CD8+ T cells. Methods: A STAT5 activator (aSTAT5) was characterized by proteomics using affinity purification mass spectrometry (AP-MS) to define its interactome and STAT5 binding specificity. STAT5 phosphorylation was assessed in hematopoietic cell lines and primary human CD8+ T cells. Results: Proteomic analysis confirmed preferential association of aSTAT5 with STAT5 relative to other proteins. In cell-based assays, aSTAT5 induced robust STAT5 phosphorylation in LCK-expressing NK-92 and Jurkat T cells, whereas phosphorylation was not observed in Raji B cells or RAW 264.7 macrophages despite expression of closely related SFKs and STAT5. Cytokine-independent STAT5 phosphorylation supported the viability of NK-92 cells and primary human CD8+ T cells during cytokine withdrawal and preserved the cytotoxic function of CAR T cells. Conclusions: We defined the interactome of a cytokine-independent STAT5 activator and demonstrated its capacity to maintain survival and function in human CD8+ T cells and NK-92 cells. These findings underscore the translational potential of engineered, cytokine-independent STAT5 activation for immune cell therapies. Full article

Chronic obstructive pulmonary disease (COPD) is a leading global cause of mortality. Its molecular pathogenesis, especially systemic immune dysregulation, remains unclear. Public transcriptomic datasets underwent machine learning to identify biomarkers, which were validated in external cohorts and single-cell RNA-seq. In vitro and ex vivo validations included: qRT-PCR and ELISA in CSE/LPS-stimulated macrophages to assess drug efficacy (Celecoxib/Lovastatin); RORC overexpression; Western blotting in patient-derived primary T cells and TCF7-deficient Jurkat cells with genetic rescue; and immunofluorescence of human lungs. A three-gene signature (RORC, TCF7, and CLEC4D) consistently discriminated COPD. CLEC4D localized to myeloid cells, while RORC/TCF7 mapped to lymphoid lineages. Celecoxib and Lovastatin attenuated macrophage inflammation, partially via RORC preservation. Crucially, the TCF7 protein was depleted in COPD primary T cells. TCF7 knockdown downregulated pro-caspase-8, which was fully reversed by TCF7 re-expression. Immunofluorescence confirmed disrupted TCF7/caspase-8 spatial patterns in COPD lungs. This signature highlights innate hyperactivation and adaptive T-cell alterations in COPD, providing novel mechanistic insights into immune dysregulation and potential pharmacological targets. Full article

Background: Genistein and resveratrol are bioactive compounds isolated from plants, recognized for their diverse biological activities including anti-cancer properties. Both compounds are also known as modulators of various types of ion channels, including voltage-gated potassium channels, Kv1.3. These channels are widely expressed in normal and cancer tissues. Their activity is crucial in regulating cell proliferation and apoptosis in cells that express Kv1.3 channels. The potential clinical application of channel inhibitors may extend to treating cancers characterized by an overexpression of these channels. Methods: This study investigates the inhibitory effects of genistein and resveratrol on Kv1.3 channels expressed in the cancer cell line Jurkat T by applying a whole-cell patch clamp. Results: Applying both compounds at concentrations ranging from 3 μM to 90 μM leads to a dose-dependent inhibition of channel activity, reducing it to approximately 50% of the control level. This inhibitory effect was reversible and associated with a significant reduction in the activation rate. When combined with simvastatin, the inhibitory effect exhibited synergy; however, it was additive when co-applied with mevastatin. Conclusions: The channel inhibition may putatively be linked to the anti-cancer activities of these compounds on Kv1.3 channel-expressing cancer cells, especially when co-applied with the statins. Full article

Chimeric antigen receptor (CAR) T-cell therapy is an effective treatment for hematologic malignancies. However, it is limited by high costs, risk of severe toxicities such as cytokine release syndrome and neurotoxicity, and heterogeneous patient responses. The current therapy monitoring depends largely on subjective symptom assessment, routine laboratory tests, and basic vital signs, without real-time, quantitative evaluation of CAR T-cell expansion or activation in clinical practice. This lack of timely immune monitoring hampers individualized care and contributes to increased treatment costs. To address this need, we present a proof-of-concept, label-free rapid optical imaging (ROI) biosensor with automated machine learning analysis for direct quantification of CAR T-cells from whole blood. This microfluidic platform integrates red blood cell (RBC) removal, CAR T-cell capture, and imaging-based quantification on a single chip, eliminating the need for centrifugation, staining, and operator-dependent interpretation. For validation, 50 μL whole blood samples spiked with Jurkat cells expressing CD19 CARs underwent RBC depletion by agglutination and microfiltration. The remaining blood components were then incubated on a sensor chip functionalized with recombinant CD19 protein. Captured CAR T-cells were imaged by brightfield microscopy and automatically enumerated using a machine learning algorithm trained on fluorescence-validated cells. The CD-19 cells’ capture performance was validated by flow cytometry and fluorescence imaging. The trained machine learning model validated at 88% sensitivity and 96% specificity. Buffer and whole blood calibration curves were established across clinically relevant concentrations (1–1000 cells/µL) with triple replicates. The results showed high correlation (0.975 and 0.990 R²) between the spiked concentration and the detected CAR T-cells, with a 95% certainty limit of detection (LOD) and quantification (LOQ) of 0.6 and 1.1 cells/µL for spiked buffer, and 14 and 67 cells/µL for spiked whole-blood, respectively. Full article