Search Results (27)

Musculoskeletal disorders are a major cause of lameness in horses, often necessitating innovative regenerative strategies to restore joint function and improve quality of life. This study investigated the effects of platelet-rich plasma (PRP), ozonized PRP, hyaluronic acid, paracetamol, and polyacrylamide hydrogel (NOLTREX^®) on the behavior of mesenchymal stem cells (MSCs) derived from equine synovial fluid. Synovial fluid samples were collected under strict cytological criteria to ensure viability, followed by in vitro expansion and phenotypic characterization of MSCs. Cultures were supplemented with the tested preparations, and cellular proliferation and viability were evaluated at 24 h, 72 h, and 7 days. PRP significantly promoted MSC proliferation in a time- and dose-dependent manner, with maximal effect at 10%. Hyaluronic acid stimulated growth, most pronounced at 1 mg/mL, while paracetamol induced a concentration-dependent proliferative response, strongest at 100 μg/mL. NOLTREX displayed a biphasic effect, initially inhibitory at high concentrations but stimulatory at 7 days. Ozonized PRP showed concentration-dependent redox activity, with lower doses maintaining viability and higher doses producing an initial suppression followed by delayed stimulation. Collectively, these findings support the therapeutic potential of PRP and related biologic preparations as intra-articular regenerative therapies in equine medicine, while underscoring the importance of dose optimization and standardized protocols to facilitate clinical translation. Full article

Human induced pluripotent stem cells (hiPSCs) hold immense promise for regenerative medicine. However, a critical barrier to the clinical application of hiPSCs is the difficulty in promoting robust cell proliferation while preserving their pluripotent state. Efficient hiPSC expansion without loss of pluripotency is crucial for generating high quality cells or therapeutic applications, disease modeling, and drug discovery. In our study, we investigated the effects of QuinoMit Q10^® fluid (QMF-Se), a nanoformulated supplement containing Ubiquinol (the active form of Coenzyme Q10) and Selenium, on hiPSC growth and maintenance in vitro. Interesting, QMF-Se supplementation significantly enhances hiPSC proliferation compared to control cultures. This increase in cell number was accompanied by heightened mitochondrial activity, suggesting improved cellular energy metabolism. Importantly, the expression of core pluripotency markers OCT4, NANOG, and SOX2 remained unaltered, confirming that the stem cells retained their undifferentiated status. Moreover, we observed that QMF-Se treatment conferred protective effects during the freeze–thaw process, reducing cell death and supporting post-thaw recovery. These results indicate that QMF-Se may improve both cell culture efficiency and cryopreservation outcomes. Overall, our findings highlight the potential of QMF-Se as a valuable additive for hiPSC culture systems, contributing to more efficient and reliable expansion protocols in regenerative medicine research. Full article

Tubastraea coccinea and T. tagusensis, commonly known as sun corals, are two species of stony corals (Scleractinia, Dendrophylliidae) native to the Indo-Pacific region (T. coccinea) and the Galapagos Islands (T. tagusensis), respectively. They are considered highly invasive species, particularly in the Western Atlantic Ocean, due to high adaptability to various ecological conditions and notable resilience. Given their demonstrated invasiveness, it is important to delve into their physiology and the molecular bases supporting their resilience. However, to date, only a few molecular tools are available for the study of these organisms. The primary objective of the present study was the development of an efficient RNA extraction protocol for Tubastraea coccinea and T.a tagusensis samples collected off Ilha Grande Bay, Rio de Janeiro (Brazil). The quantity of isolated RNA was evaluated using NanoDrop, while its purity and quality were determined by evaluating the A260/A280 and A260/230 ratios. Subsequently, based on genes known for T. coccinea, two housekeeping genes and seven stress response-related genes were isolated and characterized, for the first time for both species, using a molecular approach. An interactomic analysis was also conducted, which revealed functional interactions among these genes. This study represents the first report on gene networks in Tubastraea spp., opening new perspectives for understanding the chemical ecology and the cellular mechanisms underlying the invasiveness of these species. The results obtained will be useful for ecological conservation purposes, contributing to the formulation of strategies to limit their further expansion. Full article

Direct stochastic optical reconstruction microscopy (dSTORM) circumvents the diffraction limit of light, emerging as a powerful superresolution technique for visualizing subcellular structures with a nanoscale resolution of 10–20 nm. Yet achieving ultrastructural resolution using dSTORM alone remains challenging, despite its advantage of requiring only minimal modifications to the imaging setup and sample preparation compared to conventional fluorescence microscopy. A recent advancement that integrates expansion microscopy (ExM), which embeds specimens in a swellable polymer gel, with dSTORM holds promise for attaining imaging resolutions below 10 nm. The combined resolution, however, is governed by the expansion factor of samples, and prior studies have primarily focused on integrations involving approximately 4-fold gel expansion, as dSTORM imaging of high-fold-expanded specimens is still technically demanding. Here, we propose a pragmatic expansion strategy—post-labeling ten-fold robust expansion microscopy (plTREx)—and outline a workflow to facilitate its compatibility with dSTORM, collectively termed plTREx-dSTORM. Specifically, this workflow enhances the mechanical stability of the expansion hydrogel and improves fluorescence signal density across both widefield and dSTORM imaging platforms. Furthermore, we optimize the re-embedding protocol to integrate hydrogel expansion with dSTORM while preventing gel shrinkage. Together, plTREx-dSTORM enables highly refined imaging capable of ultrastructural interpretation of cellular proteins, effectively bridging the resolution gap between electron microscopy and optical microscopy. Full article

Gamma delta (γδ) T cells are a unique subset of T lymphocytes with distinctive features that make them highly promising candidates for cancer therapy. Their MHC-independent recognition of tumor antigens, ability to mediate direct cytotoxicity, and role in modulating the tumor microenvironment position them as versatile agents in cancer immunotherapy. This review integrates and synthesizes the existing data on γδ T cells, with an emphasis on the development and optimization of in vitro expansion protocols. Critical aspects are detailed such as activation strategies, co-culture systems, cytokine use, and other parameters to ensure robust cell proliferation and functionality, which may be valuable for those developing or optimizing clinical practices. Finally, we discuss current advancements in γδ T cell research, clinical experience, and highlight areas needing further exploration. Considering these data, we hypothesize and propose potential new applications such as engineering γδ T cells for enhanced resistance to immune checkpoint pathways or for localized cytokine delivery within the tumor microenvironment, which could broaden their therapeutic impact in the treatment of cancer and beyond. Full article

Background/Objectives: Despite the introduction of innovative therapeutics, lung cancer is still the leading cause of cancer-related death. For this reason, lung cancer still requires deep characterization to identify cellular and molecular targets that can be used to develop novel therapeutic strategies. Three-dimensional cellular models, including patient-derived organoids (PDOs), represent useful tools to study lung cancer biology and may be employed in the future as predictive tools in therapeutic decisions. However, the successful establishment of lung cancer organoids cultures that faithfully represent the respective patient tissues is still challenging due to low success rate and/or overgrowth of normal airway epithelial cells. Methods: We set up a two-step protocol that allows for establishing both short-term and long-term 3D cultures, with different characteristics and success rates. Results: Cancer tissue-originated spheroids (CTOSs) show a 100% success rate and allow for the concomitant isolation of autologous tumor infiltrating leukocytes (TILs). On the contrary, PDOs can be expanded for a medium-long term and bio-banked but retain a lower success rate and the possibility of contamination with normal airway epithelial cells. To overcome these problems, we set up an optimal medium formulation and we implemented rigorous quality controls, leading to a substantial improvement in the success rate of tumoral PDO establishment. Conclusions: Overall, this protocol guarantees flexibility and reliability, also providing useful guidelines for quality control checks to support different experimental settings. The setting up of a robust protocol for lung cancer PDO culture establishment and expansion is a key requirement for their employment both in cancer research and as predictive tools in clinical practice. Full article

Expansion microscopy has recently emerged as an alternative technique for achieving high-resolution imaging of biological structures. Improvements in resolution are achieved by physically expanding samples through embedding in a swellable hydrogel before microscopy. However, expansion microscopy has been rarely used in the field of virology. Here, we evaluate and characterize the ultrastructure expansion microscopy (U-ExM) protocol, which facilitates approximately four-fold sample expansion, enabling the visualization of different post-entry stages of the HIV-1 life cycle, focusing on nuclear events. Our findings demonstrate that U-ExM provides robust sample expansion and preservation across different cell types, including cell-culture-adapted and primary CD4+ T-cells as well as monocyte-derived macrophages, which are known HIV-1 reservoirs. Notably, cellular targets such as nuclear bodies and the chromatin landscape remain well preserved after expansion, allowing for detailed investigation of HIV-1–cell interactions at high resolution. Our data indicate that morphologically distinct HIV-1 capsid assemblies can be differentiated within the nuclei of infected cells and that U-ExM enables detection of targets that are masked in commonly used immunofluorescence protocols. In conclusion, we advocate for U-ExM as a valuable new tool for studying virus–host interactions with enhanced spatial resolution. Full article

Adipose-derived stem cells (ADSCs) hold promise for tendon repair, even if their tenogenic plasticity and underlying mechanisms remain only partially understood, particularly in cells derived from the ovine animal model. This study aimed to characterize oADSCs during in vitro expansion to validate their phenotypic properties pre-transplantation. Moreover, their tenogenic potential was assessed using two in vitro-validated approaches: (1) teno-inductive conditioned media (CM) derived from a co-culture between ovine amniotic stem cells and fetal tendon explants, and (2) short- (48 h) and long-term (14 days) seeding on highly aligned PLGA (ha-PLGA) electrospun scaffold. Our findings indicate that oADSCs can be expanded without senescence and can maintain the expression of stemness (Sox2, Oct4, Nanog) and mesenchymal (CD29, CD166, CD44, CD90) markers while remaining negative for hematopoietic (CD31, CD45) and MHC-II antigens. Of note, oADSCs’ tendon differentiation potential greatly depended on the in vitro strategy. oADSCs exposed to CM significantly upregulated tendon-related genes (COL1, TNMD, THBS4) but failed to accumulate TNMD protein at 14 days of culture. Conversely, oADSCs seeded on ha-PLGA fleeces quickly upregulated the tendon-related genes (48 h) and in 14 days accumulated high levels of the TNMD protein into the cytoplasm of ADSCs, displaying a tenocyte-like morphology. This mechano-sensing cellular response involved a complete SOX9 downregulation accompanied by YAP activation, highlighting the efficacy of biophysical stimuli in promoting tenogenic differentiation. These findings underscore oADSCs’ long-term self-renewal and tendon differentiative potential, thus opening their use in a preclinical setting to develop innovative stem cell-based and tissue engineering protocols for tendon regeneration, applied to the veterinary field. Full article

Background: Mesenchymal stem cells (MSCs) are increasingly trialed in cellular therapy applications in humans. They can also be applied to treat a range of diseases in animals, particularly in cattle to combat inflammatory conditions and aging-associated degenerative disorders. We sought to demonstrate the feasibility of obtaining MSCs from adipose tissue and characterizing them using established assays. Methods: Bovine adipose MSCs (BvAdMSCs) were isolated using in-house optimized tissue digestion protocols and characterized by performing a colony formation assay, cell growth assessments, cell surface marker analysis by immunocytochemistry and flow cytometry, osteogenic and adipogenic differentiation, and secretion of indoleamine 2,3-dioxygenease (IDO). Results: Our results demonstrate the feasibility of successful MSC isolation and culture expansion from bovine adipose tissues with characteristic features of colony formation, in vitro multilineage differentiation into osteogenic and adipogenic lineages, and cell surface marker expression of CD105, CD73, CD90, CD44, and CD166 with negative expression of CD45. BvAdMSCs secreted significant amounts of IDO with or without interferon–gamma stimulation, indicating ability for immunomodulation. Conclusions: We report a viable approach to obtaining autologous adipose-derived MSCs that can be applied as potential adjuvant cell therapy for tissue repair and regeneration in cattle. Our methodology can be utilized by veterinary cell therapy labs for preparing MSCs for disease management in cattle. Full article

Background: Regulatory T cell (Treg) therapy is considered an alternative approach to induce tolerance in transplantation. If successful, this therapy may have implications on immunosuppression minimization/withdrawal to reduce drug-induced toxicity in patients. The aim of this study was to assess the efficacy of the mTORC1/C2 inhibitor, AZD8055, in the manufacturing of clinically competent Treg cells and compare the effects with those induced by rapamycin (RAPA), another mTOR inhibitor commonly used in Treg expansion protocols. Methods: Primary human Treg cells were isolated from leukapheresis product. Cell viability, expansion rates, suppressive function, autophagy, mitochondrial unfolded protein response (mitoUPR), and cell metabolic profile were assessed. Results: We observed a stronger inhibition of the mTORC2 signaling pathway and downstream events triggered by Interleukin 2 (IL2)-receptor in AZD8055-treated cells compared with those treated with RAPA. AZD8055 induced progressive metabolic changes in mitochondrial respiration and glycolytic pathways that disrupted the long-term expansion and suppressive function of Tregs. Unlike RAPA, AZD8055 treatment impaired autophagy and enhanced the mitoUPR cell stress response pathway. Conclusions: A distinct pattern of mTOR inhibition by AZD, compared with RAPA, induced mitochondrial stress response and dysfunction, impaired autophagy, and disrupted cellular bioenergetics, resulting in the loss of proliferative potential and suppressive function of Treg cells. Full article

Ex vivo-produced dendritic cells (DCs) constitute the core of active cellular immunotherapy (ACI) for cancer treatment. After many disappointments in clinical trials, the current protocols for their preparation are attempting to boost their therapeutic efficacy by enhancing their functionality towards Th1 response and capability to induce the expansion of cytotoxic tumor-specific CD8⁺ T cells. LL-37 is an antimicrobial peptide with strong immunomodulatory potential. This potential was previously found to either enhance or suppress the desired anti-tumor DC functionality when used at different phases of their ex vivo production. In this work, we show that LL-37 can be implemented during the whole process of DC production in a way that allows LL-37 to enhance the anti-tumor functionality of produced DCs. We found that the supplementation of LL-37 during the differentiation of monocyte-derived DCs showed only a tendency to enhance their in vitro-induced lymphocyte enrichment with CD8⁺ T cells. The supplementation of LL-37 also during the process of DC antigen loading (pulsation) and maturation significantly enhanced the cell culture enrichment with CD8⁺ T cells. Moreover, this enrichment was also associated with the downregulated expression of PD-1 in CD8⁺ T cells, significantly higher frequency of tumor cell-reactive CD8⁺ T cells, and superior in vitro cytotoxicity against tumor cells. These data showed that LL-37 implementation into the whole process of the ex vivo production of DCs could significantly boost their anti-tumor performance in ACI. Full article

Gamma/delta (γδ) T cells are innate-like immune effectors that are a critical component linking innate and adaptive immune responses. They are recognized for their contribution to tumor surveillance and fight against infectious diseases. γδ T cells are excellent candidates for cellular immunotherapy due to their unique properties to recognize and destroy tumors or infected cells. They do not depend on the recognition of a single antigen but rather a broad-spectrum of diverse ligands through expression of various cytotoxic receptors. In this manuscript, we review major characteristics of the most abundant circulating γδ subpopulation, Vδ2 T cells, their immunotherapeutic potential, recent advances in expansion protocols, their preclinical and clinical applications for several infectious diseases and malignancies, and how additional modulation could enhance their therapeutic potential. Full article

Immunotherapy with chimeric antigen receptor-engineered T cells (CAR-T) has revolutionized the treatment landscape of relapsed/refractory B-cell malignancies. Nonetheless, the use of autologous T cells has certain limitations, including the variable quality and quantity of collected effector T cells, extended time of cell processing, limited number of available CAR cells, toxicities, and a high cost. Thanks to their powerful cytotoxic capabilities, with proven antitumor effects in both haploidentical hematopoietic stem cell transplantation and adoptive cell therapy against solid tumors and hematological malignancies, Natural Killer cells could be a promising alternative. Different sources of NK cells can be used, including cellular lines, cord blood, peripheral blood, and induced pluripotent stem cells. Their biggest advantage is the possibility of using them in an allogeneic context without major toxic side effects. However, the majority of the reports on CAR-NK cells concern preclinical or early clinical trials. Indeed, NK cells might be more difficult to engineer, and the optimization and standardization of expansion and transfection protocols need to be defined. Furthermore, their short persistence after infusion is also a major setback. However, with recent advances in manufacturing engineered CAR-NK cells exploiting their cytolytic capacities, antibody-dependent cellular cytotoxicity (ADCC), and cytokine production, “off-the-shelf” allogeneic CAR-NK cells can provide a great potential in cancer treatments. Full article

With the emergence of networked devices, from the Internet of Things (IoT) nodes and cellular phones to vehicles connected to the Internet, there has been an ever-growing expansion of attack surfaces in the Internet of Vehicles (IoV). In the past decade, there has been a rapid growth in the automotive industry as network-enabled and electronic devices are now integral parts of vehicular ecosystems. These include the development of automobile technologies, namely, Connected and Autonomous Vehicles (CAV) and electric vehicles. Attacks on IoV may lead to malfunctioning of Electronic Control Unit (ECU), brakes, control steering issues, and door lock issues that can be fatal in CAV. To mitigate these risks, there is need for a lightweight model to identify attacks on vehicular systems. In this article, an efficient model of an Intrusion Detection System (IDS) is developed to detect anomalies in the vehicular system. The dataset used in this study is an In-Vehicle Network (IVN) communication protocol, i.e., Control Area Network (CAN) dataset generated in a real-time environment. The model classifies different types of attacks on vehicles into reconnaissance, Denial of Service (DoS), and fuzzing attacks. Experimentation with performance metrics of accuracy, precision, recall, and F-1 score are compared across a variety of classification models. The results demonstrate that the proposed model outperforms other classification models. Full article

HCMV drives complex and multiple cellular immune responses, which causes a persistent immune imprint in hosts. This study aimed to achieve both a quantitative determination of the frequency for various anti-HCMV immune cell subsets, including CD8 T, γδT, NK cells, and a qualitative analysis of their phenotype. To map the various anti-HCMV cellular responses, we used a combination of three HLA_peptide tetramer complexes (HLA-E_VMAPRTLIL, HLA-E_VMAPRSLLL, and HLA-A2_NLVPMVATV) and antibodies for 18 surface markers (CD3, CD4, CD8, CD16, CD19, CD45RA, CD56, CD57, CD158, NKG2A, NKG2C, CCR7, TCRγδ, TCRγδ2, CX3CR1, KLRG1, 2B4, and PD-1) in a 20-color spectral flow cytometry analysis. This immunostaining protocol was applied to PBMCs isolated from HCMV⁻ and HCMV⁺ individuals. Our workflow allows the efficient determination of events featuring HCMV infection such as CD4/CD8 ratio, CD8 inflation and differentiation, HCMV peptide-specific HLA-E_UL40 and HLA-A2_pp65CD8 T cells, and expansion of γδT and NK subsets including δ2⁻γT and memory-like NKG2C⁺CD57⁺ NK cells. Each subset can be further characterized by the expression of 2B4, PD-1, KLRG1, CD45RA, CCR7, CD158, and NKG2A to achieve a fine-tuned mapping of HCMV immune responses. This assay should be useful for the analysis and monitoring of T-and NK cell responses to HCMV infection or vaccines. Full article