Search Results (5,090)

The plants from the Ocimum genus, belonging to the Labiatae family, serve as important bioresources of essential oils (EOs) rich in biologically active secondary metabolites, widely used in medicine, food, and cosmetics. This study explored the volatile composition, enantiomeric distribution, and in vitro biological activities of EOs from three Ocimum species native to Nepal: O. tenuiflorum L., O. basilicum L., and O. americanum L. EOs were extracted via hydro-distillation and analyzed using gas chromatography–mass spectrometry (GC-MS) for chemical profiling and chiral GC-MS for enantiomeric composition. Hierarchical cluster analysis was performed for major chemotypes. Antioxidant activity was assessed using DPPH and ABTS assays. Antimicrobial efficacy was evaluated using the microbroth dilution method, and cytotoxicity was tested on NIH-3T3 (normal) and MCF-7 (breast cancer) cell lines via the Cell Counting Kit-8 assay. EO yield was highest in O. tenuiflorum (1.67 ± 0.13%) during autumn and lowest in O. americanum (0.35 ± 0.02%) during winter among all Ocimum spp. The major compounds identified in O. tenuiflorum were eugenol (32.15–34.95%), trans-β-elemene (29.08–32.85%), and β–caryophyllene (19.85–21.64%). In O. americanum, the major constituents included camphor (51.33–65.88%), linalool (9.72–9.91%), germacrene D (7.75–1.83%), and β–caryophyllene (6.35–3.97%). For O. basicilum, EO was mainly composed of methyl chavicol (62.16–64.42%) and linalool (26.92–27.05%). The oxygenated monoterpenes were a dominant class of terpenes in the EOs except for O. tenuiflorum (sesquiterpene hydrocarbon). A hierarchical cluster analysis based on the compositions of EOs revealed at least three different chemotypes in Ocimum species. Chiral GC-MS analysis revealed β-caryophyllene and germacrene D as enantiomerically pure, with linalool consistently dominant in its levorotatory form. O. tenuiflorum exhibited the strongest antimicrobial activity, particularly against Candida albicans, and showed notable anticancer activity against MCF-7 cells (IC₅₀ = 23.43 µg/mL), with lower toxicity to normal cells. It also demonstrated the highest antioxidant activity (DPPH IC₅₀ = 69.23 ± 0.10 µg/mL; ABTS IC₅₀ = 9.05 ± 0.24 µg/mL). The EOs from Ocimum species possess significant antioxidant, antimicrobial, and cytotoxic properties, especially O. tenuiflorum. These findings support their potential application as natural agents in medicine, food, and cosmetics, warranting further validation. Full article

Background: Distinguishing chronic obstructive pulmonary disease (COPD) from asthma–COPD overlap (ACO) remains challenging due to shared clinical and inflammatory features. Interleukin-13 (IL-13) is implicated in airway inflammation and remodeling and may represent a potential treatable trait. This study aimed to evaluate whether serum IL-13 could differentiate between COPD and ACO or define ACO subtypes and to explore its relationship with clinical and phenotype parameters. Materials and Methods: We conducted a cross-sectional bicentric study in 215 COPD and ACO patients recruited from outpatient clinics. The study measured blood IL-13 levels in COPD vs. ACO patients, across five ACO subtypes, and evaluated IL-13’s ability to predict ACO. Additionally, correlations were explored among endotype (IL-13) and different phenotype traits (e.g., fractional exhaled nitric oxide (FeNO), sputum eosinophilia, serum total immunoglobulin E (tIgE) levels, blood eosinophilia, and neutrophilia) and clinical outcomes (annualized exacerbation rate, symptom scores, and pulmonary function parameters). Results: No significant differences in IL-13 levels were found between COPD and ACO patients or among ACO subtypes. IL-13 did not predict ACO occurrence. We observed a weak correlation between IL-13 and tIgE levels in the entire cohort. Additionally, there was a weak correlation between IL-13 and FeNO in patients with eosinophil counts exceeding 300 cells/μL, as well as between IL-13 and age in the COPD cohort. No correlation was found between IL-13 and other phenotypic features or clinical outcomes in the overall cohort, including within both COPD and ACO groups. Conclusions: IL-13 cannot differentiate between COPD and ACO or ACO’s subtypes. Full article

Perfluorooctane sulfonate (PFOS), a member of the per- and polyfluoroalkyl substance (PFAS) family, has been associated with adverse health effects, including potential links to autism spectrum disorder (ASD). This study investigates the impact of PFOS on metabolic, immunologic and behavioral profiles in BTBR-mt^B6 mice, a mouse strain that models ASD, to provide insights into the role of PFOS in ASD development and related health concerns. Three-month-old male and female BTBR-mt^B6 mice were divided into two groups (n = 6) and received daily administration of either 1 mg/kg PFOS or vehicle over a three-month period by gavage. Metabolic assessments included measurements of body weight and weekly blood glucose levels, glucose and insulin tolerance tests, organ weights, and body compositions (free fluid, fat and lean tissue). Immune profiling was conducted via flow cytometric analysis of splenic leukocytes, while behavioral evaluations included grooming, sniffing, and three-chamber social interaction tests. PFOS exposure disrupted glucose homeostasis, with both sexes exhibiting elevated blood glucose levels. Male mice showed impaired glucose tolerance, delayed glucose level recovery, and increased insulin resistance, while females displayed decreased insulin resistance. Additionally, PFOS exposure led to liver enlargement in both sexes. Behavioral assessments revealed heightened grooming in PFOS-treated males, commonly interpreted as stress- or ASD-related repetitive behaviors, whereas females exhibited reduced grooming, reflecting altered behavioral responses to exposure. Immune alterations were also sex specific. PFOS-treated males exhibited decreased granulocytes, increased macrophages, and enhanced surface expressions of B220 and CD40L. PFOS-treated females showed increased macrophages, B-cells, cytotoxic T-cells and CD25⁺ T-cell subsets, with enhanced surface expression of B220 and CD8, and reduced surface expression of Mac-3. In addition, PFOS exposure reduced spleen weight in females. Taken together, PFOS exposure induced significant physiological and behavioral changes in BTBR-mt^B6 mice, with sex-specific differences observed. These results raise concern that PFASs may contribute to the development or exacerbation of metabolic, immune and neurodevelopmental disorders, highlighting the need for sex-specific human risk assessment in environmental toxicology. Full article

Background: Type 1 diabetes (T1D) is an autoimmune disorder characterized by destruction of insulin-producing β-cells. While conventional insulin therapy manages hyperglycemia, it fails to halt autoimmunity. Oral immunotherapy targeting autoantigens like GAD65 offers potential for antigen-specific tolerance; however, its efficacy is limited by gastrointestinal degradation and poor mucosal uptake. Lactococcus lactis (L. lactis), a food-grade delivery vector, enables sustained antigen release and intestinal tract immune modulation, yet the differential transcriptomic mechanisms underlying mucosal versus systemic immune responses remain uncharacterized. Methods: Non-obese diabetic (NOD) mice were randomized into control and GAD65 groups, receiving oral PBS or the GAD65 recombinant L. lactis vaccine, respectively. Fasting blood glucose was monitored weekly. GAD65-specific IgA and IgG, along with immune tolerance-related factors, were quantified using ELISA. Lymphocyte subsets were analyzed by flow cytometry, alongside RNA sequencing and transcriptional profiling. Results: The study demonstrated that the orally administered GAD65-L. lactis vaccine could significantly induce GAD65-specific IgA antibody and TGF-β cytokine and alleviate hyperglycemia and diabetes symptoms in NOD mice. Our study facilitated the induction of GAD65-specific regulatory T cells within both intestinal lamina propria lymphocytes (LPLs) and splenic lymphocytes. Notably, antigen-specific tolerance was mainly observed in intestinal LPLs. Crucially, the immune responses elicited by the vaccine demonstrated significant disparities between intestinal LPLs and splenic lymphocytes, with intestinal LPLs exhibiting unique local immune tolerance transcriptomic profiles. Conclusions: Our findings have enhanced the comprehension of the mechanisms by which oral vaccines influence the interplay between mucosal and systemic immune responses, thereby establishing a foundational framework for the design of oral vaccines. This understanding is instrumental in advancing antigen-specific immune tolerance strategies for autoimmune diseases such as Type 1 Diabetes (T1D). Full article

Reasonable development and utilization of brackish-water resources can alleviate the pressure of freshwater scarcity in dryland areas and safeguard crop growth, but there are significant differences in brackish-water ions in different regions. Thus, exploring the mechanism of brackish-water irrigation considering brackish-water ionic differences on the growth and development of saline and alkaline dryland crops has an important production guidance value. In this study, the ionic differences in irrigated brackish water were characterized by sodium adsorption ratio using under-membrane drip-irrigated cotton as the research object, and three levels of mineralized irrigation water were designed, which were 3 g·L⁻¹ (T3), 5 g·L⁻¹ (T5), and 7 g·L⁻¹ (T7), respectively. Three different levels of sodium adsorption ratio (SAR) were set under each level of mineralization, which were 10 (mmol·L⁻¹)¹/² (S10), 15 (mmol·L⁻¹)¹/² (S15), and 20 (mmol·L⁻¹)¹/² (S20). The local freshwater irrigation was used as a control treatment. The results showed that brackish-water irrigation increased soil salt accumulation and soil water content, induced oxidative damage and disruption of ionic homeostasis in the cells, and decreased leaf photosynthetic rate. Brackish-water irrigation also significantly reduced dry matter mass by 11.04–50.12%. Reduced irrigation water SAR (S10 and S15) enhanced antioxidant enzyme activities such as superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT), and reduced malondialdehyde (MDA) content by 14.29% and 9.09%, respectively, compared with high irrigation water SAR (S20). Leaf K⁺ uptake was increased by 5.29% and 1.57% in S10 and S15, respectively, compared with S20, while Na⁺ uptake was significantly suppressed. The K⁺/Na⁺ ratio increased by 45.07%, which resulted in improved leaf photosynthetic efficiency by 25.25% and 11.91%, and significantly enhanced dry matter accumulation by 24.81% and 11.20%, respectively. In addition, compared with T3S20, the T5S10 treatment reduced the irrigation water SAR. It contributed to a significant increase in SOD, POD, and CAT activities by 30.42%, 60.70%, and 99.20%, respectively, and in plant K⁺ content and K⁺/Na⁺ by 2.48% and 38.85%, respectively, although the irrigation water mineralization increased by 66.67%. Reducing SAR could enhance photosynthesis and dry matter accumulation through the dual regulation of “antioxidant damage + ion homeostasis” in salt-stressed cotton, laying a foundation for the realization of stable and high yields of cotton under brackish-water irrigation, and providing a new perspective for the management of brackish-water resources and the sustainable development of agriculture in Xinjiang and other arid regions. Full article

Polystyrene microplastic (MP) and its co-existing contaminants may exert different toxic effects on its surrounding aquatic organisms. In order to detect the intestinal harmful responses, tilapia were subjected to exposure with 75 nm of MPs, 100 ng·L⁻¹ of sulfamethoxazole (SMZ), 5 ng·L⁻¹ of BDE153, and combinations thereof over periods of 2, 4, and 8 days. Enzymatic assays, transcriptomics, proteomics, and metabolomics were employed to evaluate intestinal histopathological effects. Results showed that significant reductions were observed in ATP, ROS, SOD, EROD, lipid metabolism-related enzymes, pro-inflammatory cytokines (TNFα and IL-1β), and apoptosis marker caspase 3 across all groups at day 8. Histological evaluation revealed diminished goblet cell density, with distinct vacuole formation in the BDE153+MPs group. KEGG pathway analysis highlighted disruptions in endocytosis, MAPK signaling, phagosome formation, and actin cytoskeleton regulation. Proteomic findings indicated notable enrichment in endocytosis (decreased sorting nexin-2; increased Si:dkey-13a21.4), MAPK/PPAR signaling, protein processing in the endoplasmic reticulum (Sec61 subunit gamma), and cytoskeletal modulation (reduced fibronectin; elevated activation peptide fragment 1), with or without SMZ and BDE153. Metabolomic profiling showed significant alterations in ABC transporters, aminoacyl-tRNA biosynthesis, protein digestion and absorption, and linoleic acid metabolism. In summary, these findings suggest that BDE153 and MPs synergistically exacerbate intestinal damage and gene/protein expression over time, while SMZ appears to exert an antagonistic, mitigating effect. Full article

In this study, oyster fermentation broth (OFB) was prepared by fermenting oysters with yeast, and its effects on oxidative stress and reproductive damage induced by tripterygium glycosides (TG) in male rats were investigated. Component analysis revealed that OFB contained bioactive substances including proteins (1.19 g/L), taurine (0.76 g/L), organic acids (2.30 mg/mL), polyphenols (123.00 mg GAE/L), flavonoids (1.97 mg RE/L), and zinc (1.10 mg/L). In vitro study revealed that OFB exhibited notable antioxidant activity, with a total antioxidant capacity of 1.28 U/mL, and DPPH, ABTS, and hydroxyl radical scavenging rates of 55.80%, 69.54%, and 48.36%, respectively. Animal experiments showed that, compared with the TG-induced model group, rats administered both low-dose (5 mL/kg) and high-dose (10 mL/kg) OFB showed significantly increased testis and seminal vesicle + prostate indices, sperm count, and serum testosterone (T) levels and decreased sperm malformation rate (p < 0.01 for all). Histological analysis of the testis revealed an increased number of spermatogenic cells and sperm within the seminiferous tubules, along with ameliorated pathological conditions compared to the model group. Potential mechanisms might be related to OFB increasing the activities of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GSH-PX) enzymes and reducing levels of malondialdehyde (MDA) in testis (p < 0.01). The findings demonstrated that OFB successfully alleviated TG-induced reproductive damage in male rats, which might be attributed to its excellent antioxidant effect. The study offers valuable insights for producing functional foods from oysters and further validates OFB’s efficacy in promoting reproductive function. Full article

Background: Neurocysticercosis (NCC) and HIV co-infection frequently occur in sub-Saharan Africa, yet the accuracy of available serological tests for NCC in immunosuppressed patients is uncertain. Methodology: We performed a cross-sectional diagnostic study on 101 people living with HIV from two endemic districts in Tanzania. Participants provided serum for cysticercosis antigen ELISA and Western Blot IgG; any positive result prompted neuroimaging investigation with cerebral computed tomography. NCC was diagnosed according to the 2017 revised Del Brutto criteria based on cCT according to Del Brutto criteria modified to exclude serology. Sensitivity, specificity, and area under the receiver–operating–characteristic curve (AUC) were calculated and adjusted for CD4 count and HIV stage. Two algorithms were compared: parallel testing (“either-test-positive”) and sequential screening (Ag ELISA screen, western blot IgG confirm). Results: NCC prevalence was 23%. Western Blot IgG outperformed Ag ELISA (sensitivity 57% vs. 30%; specificity 87% vs. 86%; AUC 0.73 vs. 0.57). Western blot IgG sensitivity declined to 54% when CD4 < 500 cells µL⁻¹, while Ag ELISA remained low. Western blot IgG positivity independently predicted NCC (adjusted odds ratio 4.1, 95% CI 1.4–11.9); Ag ELISA did not. When we counted a positive if either test was positive (parallel rule), sensitivity rose to 78% and NPV to 87%. When we ran Ag ELISA only if IgG was negative (sequential rule), we saved 70% of IgG strips, kept specificity at 95%, and PPV at 69%, but sensitivity fell to 39%. Conclusions: Western blot IgG is the most reliable single serological test for NCC in PLHIV. Parallel testing increased sensitivity and NPV and may suit better primary-level facilities without routine imaging. Sequential testing achieved high specificity, PPV, and conserved test kits, making it ideal for centers with limited reagents or scanner access. Tiered use of these assays can streamline NCC diagnosis in T. solium endemic, resource-limited settings. Full article

A current problem is the increase in skin damage, including cancer, caused mainly by prolonged exposure to ultraviolet rays from sunlight. Therefore, the aim of this work was to study the photoprotective effect to ultraviolet radiation of phenolics and acetogenic-rich extracts obtained from Annona muricata leaves applied to the skin of rats by means of gene expression in P53 and Rb, involved in tumor processes due to cell damage, in addition to the content of phenols, acetogenins and antioxidant activity present in the extract, which presented a total phenol content of 61.5 mg EAG/100 g of dry sample and flavonoids of 50 mg EQ/100 g. HPLC analysis revealed that the major compound was shikimic acid, followed by gallocatechin and 13 other phenols. DPPH analysis showed an inhibition of 64.37% and FRAP showed a value of 28,880 µmol Eq trolox/mL. The presence of acetogenins was verified by Kedde’s reagent in HPTLC. Histopathological findings in the treated groups (T4, T5) suggest thickening of the epidermis, which could be due to fibroblast proliferation. The results show a higher increase in P53 and Rb gene expression with the tested extracts compared to the positive control group, so it can be concluded that the extracts have positive effects. Full article

Sparkling wine production involves secondary alcoholic fermentation, during which carbon dioxide is trapped, creating effervescence and enhancing sensory complexity. This study evaluated the impact of Torulaspora delbrueckii and Lachancea thermotolerans yeast species using free and immobilized cells in secondary fermentation of sparkling wine, in comparison with Saccharomyces cerevisiae. Immobilized S. cerevisiae enabled faster refermentation compared to free cells, while immobilization resulted in a slower process in non-Saccharomyces strains. Biomass monitoring showed stable viable cells for immobilized S. cerevisiae during fermentation, while non-Saccharomyces strains showed a consistent reduction. Volatile profiles were positively influenced by immobilization using S. cerevisiae strains, which produced a constant increase in key aroma compounds, such as geraniol and ethyl acetate, throughout fermentation. Non-Saccharomyces strains contributed to enhanced fruity and floral aromas with variations in volatiles during refermentation. Sparkling wines fermented with immobilized L. thermotolerans were noted for ripe fruit aromas, while T. delbrueckii increased floral notes. S. cerevisiae fermentations showed higher acidity and balanced structure. These findings highlight the influence of yeast species and the yeast immobilization procedures in secondary fermentation, modulating fermentation dynamics and aroma development, and offer a promising strategy to tailor sparkling wine quality and sensory complexity. Full article

Type I collagen hydrogels are widely employed as scaffolds in tissue engineering due to their biocompatibility and ability to mimic the extracellular matrix (ECM). ECM viscoelasticity plays a critical role in regulating key cellular functions such as adhesion, proliferation, and differentiation. This study evaluates how collagen source and quality influence hydrogel architecture, mechanical properties, and keratinocyte behavior. Hydrogels were prepared at a concentration of 2.3 mg/mL using collagen from Advanced Biomatrix (AB, GLP grade) and Collagen Solutions (CS, GMP grade), and assessed for fibrillogenesis, rheological performance, and their ability to support stratified HaCaT keratinocyte cultures. AB-derived hydrogels exhibited higher porosity but lower mechanical resilience, characterized by a linear viscoelastic region (LVER) of 2.54%. In contrast, CS-derived hydrogels showed reduced porosity, denser fiber networks, and a higher LVER of 9.96%, indicating enhanced strain tolerance. HaCaT cells cultured on AB hydrogels showed diminished proliferation, metabolic activity, stratification, and expression of differentiation markers compared to those on CS hydrogels, which supported a more robust epidermal architecture. These findings highlight the critical role of collagen quality and mechanical characteristics on scaffold performance and epidermal tissue formation, emphasizing the need to optimize biomaterial properties for effective regenerative outcomes. Full article

The breast microbiome remains stable throughout a woman’s life. The breast is not a sterile organ, and its microbiota exhibits a distinct composition compared to other body sites. The breast microbiome is a community characterized by an abundance of Proteobacteria and Firmicutes, which represent the result of host microbial adaptation to the fatty acid environment in the tissue. The breast microbiome demonstrates dynamic adaptability during lactation, responding to maternal physiological changes and infant interactions. This microbial plasticity modulates local immune responses, maintains epithelial integrity, and supports tissue homeostasis, thereby influencing both breast health and milk composition. Disruptions in this balance, the dysbiosis, are closely linked to inflammatory breast conditions such as mastitis. Risk factors for breast cancer (BC) include genetic mutations, late menopause, obesity, estrogen metabolism, and alterations in gut microbial diversity. Gut microbiota can increase estrogen bioavailability by deconjugating estrogen-glucuronide moieties. Perturbations of this set of bacterial genes and metabolites, called the estrobolome, increases circulating estrogens and the risk of BC. Fusobacterium nucleatum has recently been associated with BC. It moves from the oral cavity to other body sites hematogenously. This review deals with the characteristics of the breast microbiome, with a focus on F. nucleatum, highlighting its dual role in promoting tumor growth and modulating immune responses. F. nucleatum acts both on the Wnt/β-catenin pathway by positively regulating MYC expression and on apoptosis by inhibiting caspase 8. Furthermore, F. nucleatum binds to TIGIT and CEACAM1, inhibiting T-cell cytotoxic activity and protecting tumor cells from immune cell attack. F. nucleatum also inhibits T-cell function through the recruitment of myeloid suppressor cells (MDSCs). These cells express PD-L1, which further reduces T-cell activation. A deeper understanding of F. nucleatum biology and its interactions with host cells and co-existing symbiotic microbiota could aid in the development of personalized anticancer therapy. Full article

Objective: Insulin resistance (IR) is a complex and multifactorial disorder that contributes to type 2 diabetes and cardiovascular disease. MicroRNAs (miRNAs) play important roles in diverse developmental and disease processes. However, the molecular mechanisms of IR are unclear. This paper aims to explore the role of miRNA in regulating IR and to elucidate the mechanisms responsible for these effects. Methods: IR models were created by feeding a high-fat diet (HFD) to mice or stimulating 3T3-L1 cells with palmitate. Twelve weeks of HFD trigger weight gain, leading to lipid accumulation and insulin resistance in mice. The expression profiles of miRNAs in adipose tissues (AT) from the HFD-induced mouse models were analyzed. The relationship between miR-221-3p and SOCS1 was determined using dual luciferase reporter gene assays. Metabolic alterations in AT were investigated by real-time PCR and Western blot. Results: miR-221-3p was significantly increased in AT. HFD-induced disturbances in glucose homeostasis were aggravated by miR-221-3p upregulation. The inhibition of miR-221-3p promoted insulin sensitivity including reduced lipid accumulation and the disruption of glucose metabolism. Of note, the 3′-UTR of SOCS1 was found to be a direct target of miR-221-3p. The SOCS1 inhibitor attenuated miR-221-3p-induced increases in IRS-1 phosphorylation, AKT phosphorylation, and GLUT4. miR-221-3p was considered to be involved in the PI3K/AKT signaling pathway, thus leading to increased insulin sensitivity and decreased IR in HFD-fed mice and 3T3-L1 adipocytes. Conclusions: The miR-221-3p/SOCS1 axis in AT plays a pivotal role in the regulation of glucose metabolism, providing a novel target for treating IR and diabetes. Full article

Pseudomonas aeruginosa is a clinically significant pathogen with high antibiotic resistance, necessitating rapid and reliable diagnostic methods. In this study, we developed a whole-cell aptamer selection method for P. aeruginosa using an Eppendorf-tube-based SELEX system, where bacterial cells were directly incubated with an ssDNA library. This configuration enhanced the recovery of bound aptamers and overcame the cell quantity limitations often encountered in microtiter-plate-based SELEX. After 10 selection rounds, six aptamer candidates were obtained and evaluated for affinity. Molecular docking analysis revealed that aptamer T1 possessed the highest target selectivity. To demonstrate diagnostic applicability, aptamer T1 was integrated into a hybrid lateral flow immunoassay (LFIA), replacing the conventional detection antibody. In this format, the AuNP–aptamer complex bound to the target bacteria and was captured by a specific antibody immobilized on the test line. The LFIA achieved a visual detection limit of 2.34 × 10² CFU/mL within 15 min, showing high specificity and suitability for point-of-care applications. This study presents the first demonstration of an aptamer–antibody hybrid LFIA for bacterial detection and highlights the potential of aptamers as low-cost, rapidly synthesized recognition elements adaptable for the detection of other infectious agents. Full article

Bovine leukemia virus (BLV) infects B cells in ruminants and causes lymphoma after an extended latency period. Previous studies have demonstrated T-cell exhaustion through the upregulation of immunoinhibitory molecules, including programmed death-ligand 1 (PD-L1) and T-cell immunoglobulin and mucin domain-3 (TIM-3), in BLV-infected cattle. However, studying T-cell exhaustion across all BLV infection stages remains challenging due to the virus’s prolonged latency in cattle. Sheep provide a valuable model, as they develop lymphoma more rapidly than cattle. This study examined PD-L1 and TIM-3 expression kinetics and T-cell function in BLV-infected sheep. During persistent infection, PD-L1 expression was correlated with BLV proviral load. TIM-3 expression increased in CD4⁺, CD8⁺, and γδTCR⁺ T cells. Functional analysis revealed that TIM-3 blockade enhanced T-cell activation markers (CD25 and CD69) in cultured PBMCs from infected sheep and increased CD69⁺IFN-γ⁺ and CD69⁺TNF-α⁺ populations, particularly among CD4⁺ T cells. Combined PD-L1 and TIM-3 blockade significantly enhanced cytokine production in both CD4⁺ and CD8⁺ T cells, while PD-L1 blockade alone showed limited effects. These findings demonstrate the effect of TIM-3 blockade in restoring immune function during chronic BLV infection, effective both alone and in combination. This study validates sheep as a valuable model for investigating immune checkpoint dynamics and evaluating immunotherapies for BLV infection and other chronic diseases. Full article