Search Results (691)

Male-factor infertility accounts for approxiamately half of all infertility cases globally, yet therapeutic options remain limited for individuals with no retrievable spermatozoa, such as those with non-obstructive azoospermia (NOA). In recent years, artificial gametogenesis has emerged as a promising avenue for fertility restoration, driven by advances in two complementary strategies: organotypic in vitro spermatogenesis (IVS), which aims to complete spermatogenesis ex vivo using native testicular tissue, and in vitro gametogenesis (IVG), which seeks to generate male gametes de novo from pluripotent or reprogrammed somatic stem cells. To evaluate the current landscape and future potential of these approaches, a narrative, semi-systematic literature search was conducted in PubMed and Scopus for the period January 2010 to February 2025. Additionally, landmark studies published prior to 2010 that contributed foundational knowledge in spermatogenesis and testicular tissue modeling were reviewed to provide historical context. This narrative review synthesizes multidisciplinary evidence from cell biology, tissue engineering, and translational medicine to benchmark IVS and IVG technologies against species-specific developmental milestones, ranging from rodent models to non-human primates and emerging human systems. Key challenges—such as the reconstitution of the blood–testis barrier, stage-specific endocrine signaling, and epigenetic reprogramming—are discussed alongside critical performance metrics of various platforms, including air–liquid interface slice cultures, three-dimensional organoids, microfluidic “testis-on-chip” devices, and stem cell-derived gametogenic protocols. Particular attention is given to clinical applicability in contexts such as NOA, oncofertility preservation in prepubertal patients, genetic syndromes, and reprocutive scenarios involving same-sex or unpartnered individuals. Safety, regulatory, and ethical considerations are critically appraised, and a translational framework is outlined that emphasizes biomimetic scaffold design, multi-omics-guided media optimization, and rigorous genomic and epigenomic quality control. While the generation of functionally mature sperm in vitro remains unachieved, converging progress in animal models and early human systems suggests that clinically revelant IVS and IVG applications are approaching feasibility, offering a paradigm shift in reproductive medicine. Full article

Fumonisin B1, deoxynivalenol (DON), and zearalenone (ZEA) are toxic secondary metabolites produced by Fusarium molds. These mycotoxins are common food and feed pollutants and represent a risk to human and animal health. Although the mycotoxins produced by this genus can cross the blood–brain barrier in many species, their effect on neuronal function remains unclear. We investigated the cell viability effects of these toxins on specified neural cell types, including mouse primary neuronal, astroglial, and mixed-cell cultures 24 or 48 h after mycotoxin administration. DON decreased cell viability in a dose-dependent manner, independent of the culture type. Fumonisin B1 was toxic in pure neuronal cultures only at high doses, but toxicity was attenuated in mixed and pure astroglial cultures. ZEA had significant effects on all culture types in 10 nM by increasing cell viability and network activity, as revealed by multi-electrode array measurements. Since ZEA is a mycoestrogen, we analyzed the effects of ZEA on the expression of estrogen receptor isotypes ERα and ERβ and the mitochondrial voltage-dependent anion channel via qRT-PCR. In neuronal and mixed cultures, ZEA administration decreased ERα expression, while in astroglial cultures, it induced the opposite effect. Thus, our results emphasize that Fusarium mycotoxins act in a cell-specific manner. Full article

In familial Alzheimer’s disease (FAD), presenilin 1 (PSEN1) E280A cholinergic-like neurons (ChLNs) induce aberrant secretion of extracellular amyloid beta (eAβ). How PSEN1 E280A ChLNs-eAβ affects microglial activity is still unknown. We obtained induced microglia-like cells (iMG) from human peripheral blood cells (hPBCs) in a 15-day differentiation process to investigate the effect of bolus addition of Aβ42, PSEN1 E280A cholinergic-like neuron (ChLN)-derived culture supernatants, and PSEN1 E280A ChLNs on wild type (WT) iMG, PSEN1 E280A iMG, and sporadic Alzheimer’s disease (SAD) iMG. We found that WT iMG cells, when challenged with non-cellular (e.g., lipopolysaccharide, LPS) or cellular (e.g., Aβ42, PSEN1 E280A ChLN-derived culture supernatants) microenvironments, closely resemble primary human microglia in terms of morphology (resembling an “amoeboid-like phenotype”), expression of surface markers (Ionized calcium-binding adapter molecule 1, IBA-1; transmembrane protein 119, TMEM119), phagocytic ability (high pHrodo™ Red E. coli BioParticles™ phagocytic activity), immune metabolism (i.e., high generation of reactive oxygen species, ROS), increase in mitochondrial membrane potential (ΔΨm), response to ATP-induced transient intracellular Ca²⁺ influx, cell polarization (cluster of differentiation 68 (CD68)/CD206 ratio: M1 phenotype), cell migration activity according to the scratch wound assay, and especially in their inflammatory response (secretion of cytokine interleukin-6, IL-6; Tumor necrosis factor alpha, TNF-α). We also found that PSEN1 E280A and SAD iMG are physiologically unresponsive to ATP-induced Ca²⁺ influx, have reduced phagocytic activity, and diminished expression of Triggering Receptor Expressed on Myeloid Cells 2 (TREM2) protein, but when co-cultured with PSEN1 E280A ChLNs, iMG shows an increase in pro-inflammatory phenotype (M1) and secretes high levels of cytokines IL-6 and TNF-α. As a result, PSEN1 E280A and SAD iMG induce apoptosis in PSEN1 E280A ChLNs as evidenced by abnormal phosphorylation of protein TAU at residue T205 and cleaved caspase 3 (CC3). Taken together, these results suggest that PSEN1 E280A ChLNs initiate a vicious cycle between damaged neurons and M1 phenotype microglia, resulting in excessive ChLN death. Our findings provide a suitable platform for the exploration of novel therapeutic approaches for the fight against FAD. Full article

Zebrafish are model organisms for drug screening owing to their transparent bodies, rapid embryonic development, and genetic similarities with humans. However, using standard polystyrene culture plates can limit the oxygen supply, potentially affecting embryo survival and the reliability of assays conducted in zebrafish. In this study, we evaluated the application of a novel, highly oxygen-permeable culture plate (InnoCell^TM) in zebrafish development and drug screening assays. Under both normal and oxygen-restricted conditions, zebrafish embryos cultured on InnoCell^TM plates exhibited significantly improved developmental parameters, including heart rate and body length, compared with those cultured on conventional polystyrene plates. The InnoCell^TM plate enabled a significant reduction in medium volume without compromising zebrafish embryo viability, thereby demonstrating its advantages, particularly in high-throughput 384-well formats. Drug screening tests using antiangiogenic receptor tyrosine kinase inhibitors (TKIs) revealed enhanced sensitivity and more pronounced biological effects in InnoCell^TM plates, as evidenced by the quantification of intersegmental blood vessels and gene expression analysis of the vascular endothelial growth factor receptor (vegfr, also known as kdrl). These results indicate that the InnoCell^TM highly oxygen-permeable plate markedly improves zebrafish-based drug screening efficiency and assay reliability, highlighting its potential for widespread application in biomedical research. Full article

Immunotherapy has emerged as a key modality in cancer treatment, yet its effectiveness varies significantly among patients, often due to the metabolic stress imposed by the tumor microenvironment. Hypoxia, a major factor in the tumor microenvironment, results from the high metabolic rate of tumor cells and inadequate vascularization, impairing immune cells’ function and potentially influencing gene expression profiles. Despite the widespread use of quantitative real-time PCR in immunological studies, to the best of our knowledge, data on reference gene stability in human peripheral blood mononuclear cells under hypoxic conditions is limited. In our study, we assessed the expression stability of commonly used reference genes (S18, HPRT, IPO8, RPL13A, SDHA, PPIA, and UBE2D2) in both non-stimulated and CD3/CD28-activated peripheral blood mononuclear cells cultured under normoxic, hypoxic (1% O₂), and chemically induced hypoxic conditions for 24 h. Analysis using four different algorithms—delta Ct, geNorm, NormFinder, and BestKeeper—identified RPL13A, S18, and SDHA as the most suitable reference genes for human peripheral blood mononuclear cells under hypoxic conditions. In contrast, IPO8 and PPIA were found to be the least suitable housekeeping genes. The study provides essential insights into the stability of reference genes in peripheral blood mononuclear cells under hypoxic conditions, a critical but understudied aspect of immunological research. Given the significant impact of hypoxia on T cell metabolism and function in the tumor microenvironment, selecting reliable reference genes is crucial for accurate gene expression analysis. Our findings will be valuable for future studies investigating hypoxia-driven metabolic reprogramming in immune cells, ultimately contributing to a better understanding of T cell responses in cancer immunotherapy. Full article

Nerve tissue damage is an unsolved problem in modern neurology and neurosurgery, which prompts the need to search for approaches to stimulate neuroprotection and regeneration of neural tissue. Earlier we have shown that the secretome of human mesenchymal stromal cells (MSCs) stimulates rat survival, reduces the severity of neurological deficits, and decreases the volume of brain damage in a hemorrhagic stroke model. A significant disadvantage of using the MSC secretome is the need to concentrate it (at least 5–10 fold) to achieve appreciable pharmacological activity. This increases the cost of obtaining clinically applicable amounts of secretome and slows down the clinical translation of this technology. Here, we created a number of genetically modified human MSC cultures, including immortalized MSCs and those with hyperexpression of brain-derived neurotrophic factor (BDNF) and urokinase-type plasminogen activator (uPA) and with suppressed expression of Von Hippel–Lindau tumor suppressor (VHL), and we evaluated the pharmacological activity of their secretomes in a model of intracerebral hemorrhage (ICH) in rats. The secretome of MSCs immortalized by hyperexpression of the catalytic subunit of human telomerase (hTERT) revealed neuroprotective activity indistinguishable from that of primary MSC cultures, yet it still required 10-fold concentration to achieve neuroprotective efficacy. The secretome of MSC culture with combined hyperexpression of BDNF and uPA and suppressed expression of Von Hippel–Lindau tumor suppressor even without additional concentration reduced the severity of neurological disorders and decreased brain lesion volume in the ICH model. The secretomes of MSCs with separate overexpression of BDNF and uPA or suppression of VHL had no such effect or, on the contrary, revealed a toxic effect in the ICH model. Presumably, this may be due to an imbalance in the representation of individual growth factors in the secretome of genetically modified MSCs, which individually may lead to undesirable effects in damaged nervous tissue, such as increased permeability of the blood–brain barrier (under the influence of pro-angiogenic factors) or neural cell apoptosis (due to an excess of neurotrophic factors). The obtained data show that genetic modification of MSC cultures can enhance or alter the therapeutic activity of their secretomes, which can be used in the creation of promising sources of biopharmaceutical substances. Full article

Exposure to arsenic, a known environmental and occupational genotoxicant, poses significant health risks. Identifying agents capable of mitigating its effects is crucial for public health. This study evaluates the protective potential of Argovit™ silver nanoparticles (AgNPs) against cytotoxic and genotoxic damage induced by sodium arsenite in ex vivo cultured human lymphocytes obtained from the whole blood of healthy donors. Lymphocytes were exposed to sodium arsenite (3.7 × 10⁻³ µg/mL) and Argovit™ AgNPs (3.6 × 10⁻³ µg/mL). The cytokinesis-block micronucleus (CBMN) assay was performed using a modified 144 h protocol to assess delayed effects across two cell cycles. Four groups were analyzed: untreated control, sodium arsenite only, AgNPs only, and sodium arsenite followed by AgNPs. Arsenite exposure increased cytotoxic and genotoxic biomarkers. In contrast, post-treatment with AgNPs significantly reduced these effects. All treatments were performed in duplicate, and data were analyzed using the Kruskal–Wallis test with Dunn’s post hoc comparison (p < 0.05). Statistical analysis confirmed the antigenotoxic and cytoprotective properties of Argovit™. These findings support its potential application as a mitigating agent in scenarios of environmental or occupational exposure to genotoxic compounds. Full article

Leptin, a hormone primarily produced by adipose tissue, regulates energy balance and appetite, while contributing significantly to obesity and cancer progression. Vasculogenic mimicry (VM) refers to the process by which aggressive tumor cells form blood vessel-like structures, enabling blood supply independent of endothelial angiogenesis. Metastasis-associated protein 1 (MTA1) facilitates tumor progression and metastasis. This study investigated the role of MTA1 in the relationship between leptin and VM in human breast cancer cells. Leptin upregulated the mRNA and protein expression of MTA1, as revealed by a quantitative real-time PCR and Western blot analysis, respectively. However, the Western blot revealed that leptin-induced MTA1 upregulation was inhibited by the leptin receptor (Ob-R) blocker, Ob-R BP, and the signal transducer and activator of the transcription 3 (STAT3) inhibitor, AG490. The overexpression of MTA1 was observed to induce VM in a three-dimensional culture assay and to upregulate the expression of VM-related proteins, as confirmed by the Western blot. Conversely, silencing MTA1 suppressed leptin-induced VM and the expression of VM-related proteins. These findings indicate that leptin regulates MTA1 expression through the Ob-R/STAT3 signaling pathway and that MTA1 serves as a crucial mediator of leptin-induced VM. Full article

Current routine high-sensitivity cardiac troponin assays are the criterion standard for the laboratory diagnosis of myocardial injury due to their high analytical sensitivity and specificity. However, in daily clinical practice, unexpectedly elevated cardiac troponin test results without an obvious clinical correlate are becoming more frequent compared with previous cardiac troponin assay generations. In these patients, myocardial injury may sometimes be undetected by imaging techniques, including cardiac magnetic resonance imaging. This has led to an increased interest in the pathophysiology of cardiac troponin release, particularly with regard to whether troponin can be released in the absence of myocardial necrosis and thereby resulting in an increase in cardiac troponin in the systemic circulation. Although there is in vitro evidence that cardiac biomarkers are released from reversibly injured cultured cardiomyocytes, there is still a lack of evidence for cardiac troponin release apart from different forms of cell death (i.e., apoptosis or necrosis) in animal experiments. Conversely, various circulating cardiac troponin forms have been identified in human blood samples using different analytical methods, raising the question of whether the cause of myocardial injury can be reliably determined by measuring specific circulating cardiac troponin forms. Preliminary clinical data suggests that testing for specific circulating troponin forms could increase the specificity of cardiac troponin for diagnosing acute myocardial infarctions caused by an acute coronary syndrome. This review aims to provide an up-to-date overview of these current cardiac troponin research topics with their potential clinical implications. Typical clinical cases illustrate how to interpret cTn in the individual patient and how to derive a correct diagnosis. Full article

Posidonia oceanica (L.) Delile, a Mediterranean seagrass, is rich in bioactive compounds with anti-inflammatory potential. While marine-derived molecules are increasingly studied, their direct effects on blood–brain barrier (BBB) integrity under inflammatory conditions remain largely unexplored. This study evaluated the ability of aqueous extracts from its green leaves (GLEs) and rhizomes (REs) to protect the BBB using a human in vitro model consisting of brain-like endothelial cells co-cultured with brain pericytes. The model was exposed to TNFα, with or without GLEs or REs. We assessed NO production, endothelial permeability, expression of IL-6, NLRP3, ICAM-1, VCAM-1, CLAUDIN-5, and VE-CADHERIN, and the localization of junctional proteins. TNFα increased NO and IL-6 release, upregulated ICAM-1, VCAM-1, and NLRP3, and impaired BBB integrity by altering junctional protein levels and distribution. Co-treatment with GLEs or REs reduced the production of NO, the expression of NLRP3 and adhesion molecules and restored tight and adherens junction integrity. IL-6 levels remained unaffected. These findings suggest that P. oceanica’s extracts may help preserve BBB function and mitigate inflammation-induced damage. While further studies are needed to assess their bioavailability and in vivo efficacy, these natural compounds represent promising candidates for developing preventive strategies against neuroinflammatory disorders. Full article

Arsenic, a potent metalloid contaminant of drinking water, is known for its ability to act as an initiator and modulator of disease in a variety of human tissues. Upon ingestion, arsenic is bio-transformed in the liver into a variety of metabolites, including arsenite. Arsenite permeates the blood–brain barrier (BBB), inducing oxidative stress that can be detrimental to brain neurons. As the primary glial cell at the BBB interface, astrocytes play a pivotal role in detoxifying xenobiotics such as arsenite via the production of the tripeptide antioxidant γ-glutamylcysteine, or glutathione (GSH). In this study, we assessed the mRNA levels of key components of the GSH synthetic pathway in astrocytes exposed to arsenite compared to vehicle controls. These components included xCT [substrate-specific light chain of the substrate importing transporter, system x_c⁻ (Sx_c⁻)], glutamate-cysteine ligase [both catalytic (GCL_C) and modifying (GCL_M) subunits], and glutathione synthetase (GS). Additionally, we analyzed protein levels of some components by Western blotting and evaluated functional activity of Sx_c⁻ using a fluorescence-based cystine uptake assay. Finally, we utilized a luminescence-based glutathione assay to determine the intracellular and extracellular GSH content in arsenite-treated cells. Arsenite significantly increased xCT, GCL_C, GCL_M, and GS mRNA levels, an effect blocked by the transcriptional inhibitor actinomycin D (ActD). A corresponding increase in Sx_c⁻ activity was also observed in the arsenite treatment groups, along with significant increases in GCL_C and GCL_M protein expression. However, no increase in GS protein expression was detected. Finally, arsenite treatment significantly increased extracellular GSH levels, an effect which was also prevented by the inclusion of ActD. Overall, our study provides evidence that arsenite transcriptionally regulates several cellular processes necessary for GSH synthesis in primary cortical astrocyte cultures, thereby contributing to a better understanding of how this environmental toxicant influences antioxidant defenses in the brain. However, these results should be interpreted with caution regarding their applicability to vivo systems. Full article

Arboviruses, transmitted by blood-sucking arthropods, are responsible for significant human and animal diseases, including fever, hemorrhagic fever, and encephalitis, posing a serious threat to global public health. Nevertheless, research on the mechanisms of arbovirus infection and the development of therapeutic interventions has been impeded. This delay is primarily due to the limitations inherent in current in vitro research models, including cell cultures and animal models. The simplicity of cell types and interspecies differences present significant obstacles to advancing our understanding of arbovirus infection mechanisms and the development of effective drugs. Human brain organoids, derived from human pluripotent stem cells or human embryonic stem cells and cultured in three-dimensional systems, more accurately replicate the extensive neuronal cellular diversity and key characteristics of human neurodevelopment. These organoids serve as an ideal model for investigating the intricate interactions between viruses and human hosts, and providing a novel platform for the development of antiviral drugs. In this review, we summarize how brain organoid models complement classical approaches to accelerate research into the infection mechanisms of arboviruses, with a particular focus on the types of neural cells, key factors, and cellular signaling pathways involved in the arbovirus infection of brain organoids that have been reported. Furthermore, we examine the development of brain organoids, address their current limitations, and propose future directions to enhance the application of brain organoids in the study of arboviral infectious diseases. Full article

Colpodella species are free-living predatory protists that prey on algae, ciliates and bodonids using myzocytosis. Colpodella species have been reported in human and animal infections. Polymerase chain reaction (PCR) using primers targeting 18S rRNA genes of Cryptosporidium and piroplasms have identified Colpodella species in arthropods, host blood and feces, demonstrating the phylogenetic closeness of Colpodella species to the apicomplexa. However, in human, animal and arthropod hosts, the life cycle stages of Colpodella are unknown. In this literature review, we provide an overview of the widespread occurrence of Colpodella species in ticks, and pathogenicity in humans and animals. We discuss methods for culture and microscopy that can aid diagnosis. Phylogenetic tree analysis of Colpodella species identified using 18S rRNA demonstrates that the Colpodella species identified in different geographic regions represent different species and strains that may impact virulence and zoonotic transmission. There is a pressing need to culture Colpodella species, and to stain cells for morphological identification. This will aid molecular investigations aimed at identifying molecular markers of Colpodella spp. facilitating transmission, survival and pathogenesis in hosts, and will determine which species and strains to prioritize for the risk of zoonotic infections to humans and for infections in animals. Full article

Background and Objectives: Neuronal nitric oxide synthase (nNOS) overexpressed in melanoma plays a critical role in disease progression. Our previous studies demonstrated that nNOS inhibitors exhibited potent anti-melanoma activity and regulated PD-L1 expressions in the presence of interferon-gamma (IFN-γ). However, the role of nNOS in the melanoma immune response has not been well defined. Methods: Changes in gene expression profiles after nNOS inhibitor treatment were determined by transcriptomic analysis. A melanoma mouse model was used to determine the effects of nNOS inhibition on peripheral T cells and the in vivo anti-tumor activity of combining nNOS inhibitors with immune checkpoint blockade. Changes in human T cell activation through interleukin-2 (IL-2) production were investigated using an ex vivo co-culture system with human melanoma cells. Results: Cellular RNA analysis revealed significant changes in the genes involved in key signaling pathways after nNOS inhibitor HH044 treatment. Immunophenotyping of mouse peripheral blood mononuclear cells (PBMCs) after prolonged HH044 treatment showed marked increases in CD4⁺ and CD8⁺PD-1⁺ T cells. Ex vivo studies demonstrated that co-culturing human PBMCs with melanoma cells inhibited T cell activation, decreasing IL-2-secreting T cells both in the presence and absence of IFN-γ. PBMCs from a significant portion of donors (7/11, 64%), however, were reactivated by nNOS inhibitor pretreatment, displaying a significant increase in IL-2⁺ T cells. Distinctive T cell characteristics were noted at baseline among the responders with increased CD4⁺RORγt⁺ and reduced CD4 naïve T cells. In vivo mouse studies demonstrated that nNOS inhibitors, when combined with PD-1 blockade, significantly reduced tumor growth more effectively than monotherapy. Additionally, the median survival was extended from 43 days in the control mice to 176.5 days in mice co-treated with HH044 and anti-PD-1. Conclusions: Targeting nNOS is a promising approach to enhancing the anti-melanoma activity of immune checkpoint inhibitors, not only interfering with melanoma biological activities but also regulating the tumor microenvironment, which subsequently affects T cell activation and tumor immune response. Full article

Understanding the function of the blood–brain barrier (BBB) in health and disease, as well as improving drug delivery across the BBB, remains a critical priority in neuroscience research. However, current in vitro models of the BBB have become increasingly complex and challenging to implement. In this study, we present a simplified microfluidic BBB model in which human umbilical vein endothelial cells (HUVECs) are cultured as a monolayer along a fibrin gel containing human pericytes and astrocytes. Remarkably, within just three days, the 3D co-culture significantly enhanced barrier formation and upregulated the expression of tight-junction proteins in HUVECs. These findings demonstrate that HUVECs, which have been extensively used for over 50 years to study vascular endothelium due to their ease of isolation and culture, can adapt their phenotype towards that of BBB endothelial cells under appropriate conditions. This microfluidic BBB model offers a valuable tool for drug development and for advancing our understanding of BBB physiology in both health and disease contexts. Full article