Search Results (14,363)

Chronic inflammatory diseases and autoimmune diseases are overlapping but distinct immune-mediated disorders that represent a growing worldwide health concern, characterised by persistent inflammation, tissue damage, and progressive organ dysfunction. In the United States alone, more than $180 billion is spent annually on managing these conditions, yet fewer than 10% of patients achieve long-term remission. These figures highlight the limitations of conventional therapies, which often control symptoms rather than adequately modify the underlying disease process. This review provides a focused and comparative overview of emerging therapeutic strategies across representative immune-mediated disorders, with particular emphasis on mesenchymal stem cells, Janus kinase-signal transducer and activator of transcription (JAK-STAT) inhibitors, chimeric antigen receptor T-cell therapies, therapeutic vaccines, microbiome-modulating interventions, and nanotechnology-based drug delivery systems. In parallel, artificial intelligence (AI) is increasingly contributing to biomarker discovery, drug repurposing, and treatment stratification, thereby supporting the development of predictive and personalised medicine. Overall, these advances support a shift toward mechanism-based, multimodal, and more durable treatment strategies, although further clinical validation remains necessary. Full article

This study reports the synthesis, characterization, DFT calculations and in vitro antimicrobial, cytotoxic and antiproliferative evaluation of La(III), Eu(III), and Gd(III) metal complexes with ampicillin. The compounds were characterized by Thermal Gravimetric Analysis (TGA), elemental analysis, ultraviolet–visible spectroscopy (UV–Vis), Fourier-transform infrared spectroscopy (FTIR), and proton nuclear magnetic resonance (¹H NMR), indicating a 2:1 metal-to-ligand ratio with ampicillin, and likely, a coordination through carbonyl, carboxylic and β-lactam groups, with the general formula [Ln₂(L)(Cl)₅(H₂O)_x] (Ln = La(III), Eu (III), Gd (III), and x = 2 for La(III), 5 for Eu(III) and Gd(III), L-ampicillin anion). Antimicrobial studies showed activity against ampicillin-resistant Staphylococcus aureus (MIC = 15.6 µg·mL⁻¹) but no activity against Escherichia coli. In cytotoxicity studies, all complexes inhibited B16-F10 (murine melanoma) proliferation, with GI₅₀ values around 140 µg·mL⁻¹. Against U251 (glioma) cell line, only [Eu₂(L)(Cl)₅(H₂O)₅] exhibited cytotoxicity activity, GI₅₀ = 104 µg·mL⁻¹, and notably, [Eu₂(L)(Cl)₅(H₂O)₅] was active against MCF7 (breast carcinoma) with a GI₅₀ = 8.1 µg·mL⁻¹. However, all complexes exhibited high cytotoxicity in NIH-3T3 cells (GI₅₀ = 0.030–2.90 µg·mL⁻¹), indicating limited selectivity between normal and cancer cells. Nevertheless, except for the La complex, most compounds were less cytotoxic than doxorubicin, highlighting the need for further optimization to improve selectivity. Full article

Low-temperature stress severely restricts the engineering application of anaerobic ammonia oxidation (anammox) technology in municipal mainstream wastewater treatment, leading to its slower large-scale implementation relative to industrial wastewater and reject water treatments. The inhibitory effects of low temperatures on the anammox process cannot be merely ascribed to conventional microbial metabolic responses. Elucidating the specific mechanisms underlying low-temperature impacts on anammox bacteria is therefore critical for formulating targeted mitigation strategies. In this study, a meta-analysis was performed to compare the response patterns of specific anammox activity (SAA) and nitrogen removal rate (NRR) to temperature variations. SAA declines gradually with decreasing temperature, while NRR displays a more dramatic and stepwise reduction. The T₅₀ values (temperature corresponding to 50% of the performance at 30 °C) for these two parameters are 20 °C and 15 °C, respectively. Low-temperature inhibition of anammox is a multifaceted process, encompassing direct physiological disturbances to individual anammox cells and impaired nitrite bioavailability within the microbial community. To address these temperature-related bottlenecks, a conceptual hybrid nitrogen removal system was rationally optimized by integrating conventional strategies with an innovative split-flow influent regulation strategy. This hybrid system is anticipated to enhance the stability and treatment efficiency of anammox under low-temperature conditions, thus facilitating its broader engineering application in cold climate regions. Full article

Glypican-3 (GPC3)-targeted chimeric antigen receptor T (CAR-T) cell therapy is a promising approach for hepatocellular carcinoma (HCC), but marked interpatient variability and antigen heterogeneity limit its broader application. Here, we established a patient-derived organoid (PDO)-based platform to functionally evaluate autologous GPC3-targeted CAR-T cell activity in HCC. HCC PDOs preserved key histologic features and heterogeneous GPC3 expression patterns of the original tumors. In co-culture assays, CAR-T cell cytotoxicity was associated with GPC3 expression levels and was accompanied by IFN-γ and IL-2 release, supporting the feasibility of using PDOs for functional assessment of CAR-T cell sensitivity. We further found that matrix conditions strongly influenced organoid architecture, viral transduction, CAR-T cell infiltration, and killing efficiency, with lower Matrigel concentrations providing a more permissive setting for functional assessment. Importantly, in GPC3-low PDOs, pretreatment with the DNA methyltransferase inhibitor 5-azacytidine (5-AZA) increased surface GPC3 expression, reduced DNA methyltransferase 3 alpha (DNMT3A) expression, and significantly enhanced CAR-T-mediated cytotoxicity. Together, these findings provide proof-of-concept evidence supporting the use of HCC PDOs as a patient-derived platform for modeling selected determinants of GPC3-targeted CAR-T cell activity and for exploring combination strategies to improve therapeutic efficacy. Full article

Background: Cardiolipin (CL) is a phospholipid composed of a glycerol linked with two phosphatidate moieties that constitutes an integral part of the human inner mitochondrial membrane under physiological conditions. It is also vital for bacterial membrane transport and key bacterial functions associated with cell division and infection. CL is released in the cytosol or into the extracellular milieu upon cell death and during inflammation. We therefore tested the ability of CL to activate and expand tumor infiltrating lymphocytes (TIL) from patients with epithelial cancer. Methods: TIL were isolated from gastrointestinal tumor tissues and expanded in vitro in the presence of CL. The role of the NLRP3 inflammasome was evaluated using the specific inhibitor MCC950 and siRNA-mediated silencing of NLRP3. Phenotypic changes and T-cell potency were assessed via CXCL9/10 expression levels. To characterize the immune repertoire, deep TCR sequencing was performed to compare the TCR Vα and Vβ CDR3 regions between TIL and the corresponding tumor tissue. Recognition of autologous tumor cells and tumor-specific mutations, including mutations in KRAS and mitochondrial UQCRFS1 (D145V), was assessed using MHC class I and II restriction assays. Results: CL-expanded TIL exhibited increased CXCL9/10 expression, which is associated with increased potency of tissue invasion. CL-TIL exhibited broader recognition of frequently occurring KRAS mutations, and this effect could be blocked with an inhibitor (MCC950) of the NLRP3 pathway, a multiprotein inflammatory complex associated with danger signaling. TIL exhibited an enriched TCR Vα and Vβ CDR3 repertoire compared to tumor tissue, as defined by deep TCR sequencing. TCR αβ+ TIL recognized autologous tumor tissue in an MHC class I– and class II–restricted fashion, including the mutant HLA-DP–restricted mitochondrial protein associated with the electron respiratory chain complex III (UQCRFS1 D145V) presented by autologous tumor cells. Conclusions: CL activates the NLRP3 inflammasome pathway in TIL from patients with GI cancer and increases CXCL9/CXCL10 expression in TIL, resulting in enhanced recognition of mutant cancer–associated target epitopes, including a mitochondrial protein. CL may provide a danger signal: that facilitates TIL expansion via CL-activated pathways. Full article

Toxoplasma gondii, an obligate intracellular protozoan infecting approximately one-third of the global population, poses a significant yet underappreciated threat to reproductive health in both sexes. Although this parasite has long been linked to birth defects caused by infection during pregnancy, new research shows that it also reduces fertility in both sexes through different but related mechanisms. This review synthesizes knowledge on T. gondii-induced reproductive pathology across females and males, examining shared mechanistic themes while respecting tissue-specific differences, and evaluates emerging therapeutic strategies. In females, the parasite establishes persistent uterine reservoirs, triggers decidual immune dysregulation characterized by NK cell cytotoxicity, M1 macrophage polarization, Treg apoptosis, and inflammasome-mediated pyroptosis, while disrupting estrogen and progesterone signaling through both host receptor modulation and intrinsic parasite steroidogenic enzymes (TgCYP450mt, TgMAPR, Tg-HSD). In males, T. gondii breaches the blood–testis barrier, induces germ cell and Leydig cell apoptosis via ER stress and caspase pathways, impairs sperm quality parameters across acute and chronic infection, and disrupts the hypothalamic–pituitary–gonadal axis. Conserved molecular mechanisms—including NLRP3 inflammasome activation, PERK/eIF2α/ATF4/CHOP-mediated ER stress, and oxidative stress—operate in both reproductive tissues. The parasite’s intrinsic steroidogenic capability and bidirectional hormonal manipulation represent a paradigm shift in understanding host–parasite interactions. Conventional antiparasitics face limitations due to poor reproductive sanctuary penetration. Immunomodulatory approaches targeting Trem2, Tim-3, and the NLRP3 inflammasome show promise, along with natural products including Inonotus obliquus polysaccharide and ginseng polysaccharide. Nanomedicine platforms and mRNA vaccine candidates offer new directions for overcoming tissue barrier limitations. Toxoplasma gondii represents a fundamental threat to fertility and pregnancy outcomes rather than merely a risk for congenital infection. Integrated therapeutic strategies addressing direct parasitism, immunopathology, and endocrine disruption are needed. Longitudinal cohort studies, strain-specific mechanistic comparisons, and clinical trials of immunomodulatory adjuncts are urgently required. Full article

Background: A major challenge in antiviral development is the identification of novel virus–host interactions while ensuring therapeutic efficacy and safety. These challenges have renewed interest in phytochemicals derived from medicinal plants as alternative antiviral agents. Objectives: In this study, we investigated the antioxidant, antiviral, and immunomodulatory properties of a Mediterranean Urtica dioica extract (UdE) against SARS-CoV-2 using chemical, biochemical, and in vitro approaches. Methods: The physicochemical properties of UdE were characterized using microtiter assays and HPLC analysis. Cytocompatibility was evaluated in HEK293T, Vero E6, Caco-2, and Calu-3 cell lines while antioxidant activity was assessed using both chemical and cell-based assays. Antiviral activity was evaluated by assessing inhibition of SARS-CoV-2 receptor binding domain (RBD)–ACE2 interaction using ELISA, inhibition of SARS-CoV-2 main protease (Mpro) activity via FRET assay and inhibition of viral entry using SARS-CoV-2 S1 pseudovirus neutralization assay. Results: UdE (100 µg/mL) inhibited RBD–ACE2 binding by 94% and suppressed Mpro activity by 74%, while reducing moderate but significant inhibition of pseudovirus entry (33.6%) at 300 µg/mL dose level in ACE2 expressing HEK293T cells. Immunomodulatory analysis revealed significant suppression of IL-1β and IL-6 production, accompanied by increased TNF-α and IL-8 levels. Conclusions: Collectively, these findings highlight that UdE exhibits multi-target in vitro antioxidant, antiviral, and immunomodulatory activity against SARS-CoV-2; therefore, UdE represents a promising bioactive extract for the management of SARS-CoV-2 infection. Full article

Background: Despite hybrid and vaccine-induced immunity, SARS-CoV-2 continues to cause disease. The characterization of humoral and cellular immune responses is essential for guiding prevention strategies and booster dose policies; Methods: A prospective cohort study was conducted with 131 hospitalized patients with confirmed COVID-19 in the Aburrá Metropolitan Valley, Colombia. Clinical and immunological data were evaluated on days 1–3, days 5–7, days 8–12, and 4–5 months after diagnosis. Humoral immunity was assessed by enzyme-linked immunosorbent assay (ELISA), chemiluminescent microparticle immunoassay (CMIA), and neutralization testing, and cellular immunity by CD4⁺/CD8 T-cell responses. Results: vaccinated patients had higher baseline levels of IgG and neutralizing antibody positivity than unvaccinated patients (ELISA 89.1% vs. 60.0%; CMIA 86.4% vs. 50.0%; neutralizing antibodies 88.2% vs. 65.0%), but cases of severe disease occurred in both groups. Adults aged ≥65 years had higher antibody positivity, but severe disease persisted. Mortality at 28 days was 7.6%, mainly among critically ill patients with comorbidities. Antibodies persisted at 4–5 months but were lower in those with severe acute disease. Those who received the booster dose showed stronger CD4⁺/CD8⁺ activation (notably against the Omicron variant) than unvaccinated/partially vaccinated patients. Conclusions: Vaccination improved humoral and cellular responses, but severe breakthrough infections still occurred, particularly in high-risk patients. Full article

Globally, the rise in MDR P. aeruginosa infections poses a serious threat to public health, as these strains frequently exhibit extensive resistance to conventional antibiotics, prompting interest in bacteriophages as alternative treatments. In this study, we isolated and characterized a lytic P. aeruginosa phage, vB_Pae_YuaWU01, from hospital sewage in southern Thailand. Morphological analysis revealed Siphovirus-like characteristics. The phage demonstrated efficient host adsorption, with approximately 85.9% of particles attached within 15 min, and exhibited a latent period of 50 min with a burst size of 17.2 PFU/cell. It showed strong lytic activity, consistently suppressing bacterial growth without no regrowth observed over 72 h. Notably, the phage significantly inhibited biofilm formation by up to 59.9% and reduced pre-established biofilms by 39.78% at the highest tested concentration (10⁹ PFU/mL). Genome analysis revealed a 61,824 bp double-stranded DNA genome with 64.48% GC content and 88 predicted genes. Bioinformatic analysis suggests that the genome is organized into structural, replication, and lysis modules. Importantly, no toxin, antimicrobial resistance, lysogeny, or tRNA genes were identified, suggesting a favorable safety profile. The phage was classified within the genus Yuavirus, showing 97.4% genomic similarity to Sphaerotilus phage SN1, which infects a different host strain. The findings highlight its potential as a genetically safe therapeutic agent; however, its limited host range indicates that it may be best positioned as a strategic component of phage cocktails or as a synergistic partner with antibiotics to maximize therapeutic efficacy. Full article

Background/Objectives: Retinoblastoma treatment remains limited by therapeutic resistance and toxicity. While melphalan is a key chemotherapeutic agent, its efficacy is constrained by adverse effects. Curcumin has emerged as a potential adjunct owing to its capacity to regulate oxidative stress and oncogenic signaling pathways, including STAT3. This study aimed to assess the synergistic tumor-inhibitory effects of melphalan–curcumin combined treatment and to investigate the roles of ROS, apoptosis, and STAT3-associated signaling, including validation in a three-dimensional (3D) tumor spheroid model. Materials and Methods: Human retinoblastoma (WERI-Rb-1) and normal keratinocyte (HaCaT) cells were exposed to melphalan, curcumin and the combined treatment regimen. Cell viability was analyzed by MTT assay, and drug interactions were analyzed using the Chou–Talalay method. Migration was evaluated by scratch assay. Intracellular ROS levels were quantified using the DCFH-DA assay and confirmed by flow cytometry. Apoptosis was quantified by Annexin V/PI staining, and caspase activity was assessed colorimetrically and by immunocytochemistry. Cytokine levels were determined by ELISA, and gene expression profiling of STAT3 and apoptosis-associated genes were performed using qRT-PCR. Three-dimensional tumor spheroids were established to evaluate treatment responses in a physiologically relevant model. The contribution of ROS was further investigated using N-acetyl-L-cysteine (NAC) pretreatment. Results: The combination of melphalan and curcumin notably reduced WERI-Rb-1 cell viability in a synergistic manner (CI < 1) while exhibiting lower cytotoxicity in HaCaT cells, indicating selective antitumor activity. Co-treatment markedly inhibited cell migration and increased intracellular ROS levels. Cells pretreated with NAC significantly reduced ROS levels accumulation and moderately restored cellular viability, supporting a contributory role of oxidative stress. The combination treatment induced pronounced apoptosis, with increased early and late apoptotic cell populations, enhanced caspase-7 and caspase-9 activity, and elevated caspase-9 protein expression. These effects were associated with upregulation of pro-apoptotic genes (BAX, CASP3, CASP7, CASP9), downregulation of anti-apoptotic genes (BCL2, SURVIVIN), and reduction in STAT3 mRNA expression. In addition, the combination reduced pro-inflammatory cytokine levels. Importantly, these effects were recapitulated in 3D tumor spheroids, where the combination treatment reduced spheroid size and viability and induced structural disruption. NAC-mediated rescue experiments in 3D models further supported the notion that ROS contributes to, but is not solely responsible for, the observed effects. Conclusions: Overall, these results suggest that melphalan and curcumin exert synergistic and selective antitumor effects in retinoblastoma cells, associated with changes consistent with ROS-related effects, mitochondrial apoptotic processes, and STAT3-related transcriptional alterations rather than definitive pathway activation. The validation of these effects in a 3D tumor spheroid model provides additional support for the potential clinical significance of this combined treatment; however, additional protein-level and functional validation is required. Full article

Background/Objectives: Histone deacetylase (HDAC) inhibitors have been shown to prime the response to immunotherapy (IMT) treatment by inducing immune activation and infiltration to target tumor cells. Many studies primarily focus on adaptive immune cells and their expression of pro-inflammatory markers, like somatostatin receptor 2 (SSTR2); however, macrophages are known to help mediate key tumor microenvironment changes. The goal of this study is to evaluate the effects of HDAC inhibitors and IMT on macrophages, their expression of SSTR2, and their impact on the treatment response in triple-negative breast cancer (TNBC). Methods: Cytotoxic effects of HDAC inhibitors on 4T1 mouse mammary carcinoma cells, including suberoylanilide hydroxamic acid (SAHA), were evaluated using flow cytometry. Bone marrow-derived macrophages (BMDMs) were stimulated to M1-like and M2-like phenotypes and treated with SAHA to explore the effects on SSTR2 expression in different macrophage phenotypes. 4T1-tumor-bearing BALB/c mice were used to evaluate the therapy response to four treatments: saline control, SAHA, anti-PD-1 + anti-CTLA-4 checkpoint blockade IMT, or a combination of SAHA + IMT. Additional cohorts of 4T1-tumor-bearing BALB/c mice and NOD SCID mice, which lack adaptive immune cells, were euthanized for early evaluation of tumor-associated macrophage (TAM) populations via flow cytometry and cytokine analysis. One-way independent ANOVAs and log-rank tests were used to compare group differences. Results: SAHA promotes SSTR2 expression on M1-like BMDMs in vitro. SAHA promotes M2-like TAMs in vivo and stimulates pro-inflammatory, anti-tumor cytokine production in combination with IMT. Conclusions: SAHA drives SSTR2 expression and anti-tumor innate immune responses with additive effects in combination with immunotherapy in preclinical TNBC. Full article

Adipogenesis is a critical factor in causing obesity, which is a global health problem associated with metabolic disorders, such as insulin resistance and cardiovascular diseases. Natural compounds with anti-adipogenic activity may represent potential approaches for modulating adipocyte function. However, despite increasing interest in natural products, the anti-adipogenic potential of acridone alkaloids, particularly prenylated derivatives, remains largely unexplored. This study examined the effects of N-methylatalaphylline (NMA), a prenylated acridone alkaloid, on adipocyte differentiation, lipid accumulation, and glucose uptake. NMA exhibited anti-adipogenesis, particularly toward preadipocytes, and significantly reduced lipid accumulation in murine 3T3-L1 and human PCS-210-010 adipocytes at nontoxic doses (1.5–6 µM). At 3–6 µM, NMA downregulated adipogenic regulators, including PPARγ, C/EBPα, and SREBP1, along with adipogenic effectors, such as FABP4, adiponectin, LPL, PLIN1, and FAS. Mechanistic studies indicated that NMA treatment was associated with reduced phosphorylation of AKT, ERK, and p38, accompanied by cell-cycle arrest and inhibition of mitotic clonal expansion. Meanwhile, activation of AMPK-ACC signaling, which may contribute to suppression of adipogenesis and reduced glucose uptake, was observed in differentiated 3T3-L1 cells after treatment with 6 µM NMA for 48 h. Additionally, molecular docking and molecular dynamics simulations suggested potential interaction between NMA and ERK1, supported by hydrogen bonding and hydrophobic contacts. Overall, these findings suggest that NMA exerts anti-adipogenic effects in vitro by modulating adipocyte proliferation, differentiation, and lipid metabolism. These findings highlight NMA as a promising acridone alkaloid scaffold for anti-adiposity applications, warranting further in vivo validation. Full article

Background/Objectives: Spontaneous remission (SR) of acute myeloid leukemia (AML) offers unique clinical insights into host anti-tumor immunity. However, the comprehensive clinical landscape and molecular dynamics of blast clearance and subsequent relapse remain unclear. This study aimed to elucidate these dynamics. Methods: We conducted a two-phase observational study: a systematic pooled analysis of 66 adult AML SR cases (1990–2024) to define clinical triggers and outcomes and longitudinal molecular tracking of two institutional cases to map clonal shifts (with immune profiling for Patient 1 and genomic tracking for both). Results: In the pooled analysis, infection was the predominant trigger, accounting for 78.6% (95% CI: 65.6–88.4%) of SR events. The dataset showed male predominance and monocytic leukemia enrichment (57.6% [95% CI: 44.1–70.4%]), suggesting lineage-specific susceptibility. SR duration and relapse risk were independent of the infection trigger, AML subtype, or age. When integrated with these clinical patterns, institutional tracking was consistent with a biphasic evolutionary model: an acute IL-8 surge alongside NKT and CD4+ T cell activation coincided with blast clearance, as observed primarily in Patient 1. Subsequently, the emergence of TP53 or NRAS mutations within persistent DNMT3A-mutated clones during relapse raised the hypothesis that unresolved chronic inflammation could potentially exert selective pressure favoring resistant subclones. Such interpretations remain correlational and require prospective validation. Conclusions: Our findings outline a clinical–evolutionary framework for AML SR. Remission durability likely relies on balancing acute immune activation with underlying clonal stability. These observational insights highlight complex immune-genomic crosstalk, generating hypotheses for future prospective investigations. Full article

Natural antioxidants are essential for protecting the body against oxidative stress and exhibit a wide range of biological activities. In this context, forty extracts derived from ten submerged cultivated mushroom species were analyzed for their mycochemical composition, antioxidant capacity, and cytotoxic effects against MCF7 breast cancer cells. Qualitative and quantitative screening revealed that, among the detected classes of bioactive compounds, the extracts were predominantly enriched in flavonoids, terpenoids, and phenolic constituents. Considerable variation was observed in the levels of total phenolics, flavonoids, and ascorbic acid among different species and solvent extracts. The highest total phenolic contents were detected in ethanol and ethyl acetate extracts of G. frondosa (110.0 ± 6.4, 227.6 ± 14.2, and 160.5 ± 5.3 mg GAE/g), while the water extract of F. velutipes also exhibited elevated phenolic levels (119.2 ± 6.5 mg GAE/g). Flavonoid concentrations ranged from 102.5 ± 10.5 to 359.9 ± 2.5 mg QE/g in biomass and culture liquid extracts obtained with organic solvents. Ascorbic acid content was generally highest in ethyl acetate culture liquid extracts, suggesting solvent-dependent enrichment of antioxidant metabolites. Free radical scavenging activity increased in a concentration-dependent manner, reaching inhibition values more than 90% at 20 mg/mL in all tested mushrooms. Cytotoxicity assays demonstrated that extract type, solvent, and incubation time strongly influenced the inhibition of MCF7 cell viability. Ethyl acetate extracts from H. erinaceus, P. ostreatus, T. versicolor, and T. pubescens exhibited the strongest cytotoxic effects, reducing cell viability by up to 70% at higher concentrations. The results demonstrate that mushroom extracts, particularly ethyl acetate extracts, possess significant antioxidant and cytotoxic activities. These findings highlight their potential as promising natural sources of medicinal bioactive compounds for antioxidant and anticancer applications. Full article

P2X7 is an adenosine 5′-triphosphate (ATP)-gated ion channel. CD39 (ectonucleotidase triphosphate diphosphohydrolase-1) hydrolyses ATP to reduce its extracellular concentration to limit P2X7 activation. Both P2X7 and CD39 are present on leukocytes. Ficoll-Paque density gradient centrifugation is widely used to isolate human peripheral blood mononuclear cells (PBMCs), yet the extent to which other density gradient centrifugation methods influence the expression of cell surface molecules remains unclear. Using mass cytometry, this study compared the proportions of mononuclear leukocyte subsets and the relative amount of cell surface P2X7 and CD39 detected on these cells in paired, bar-coded cryopreserved human PBMC samples isolated by Ficoll-Paque or SepMate Tube density gradient centrifugation. Both techniques yielded similar proportions of mononuclear leukocyte subsets. P2X7 and CD39 were detected across all cell subsets, with the relative amount of P2X7 or CD39 comparable between separation methods. Relatively minor but statistically significant differences were observed for some populations. P2X7 expression was higher on CD3⁺, CD4⁺, and conventional CD4⁺ T cells, and naïve B cells, and lower on myeloid dendritic cells, while CD39 expression was lower on regulatory T cells in SepMate Tube samples compared to Ficoll-Paque samples. Overall, Ficoll-Paque and SepMate Tube density gradient centrifugation yield comparable results within PBMC samples, supporting the use of either method in studies examining immune phenotypes including the purinergic molecules P2X7 and CD39. Full article