Search Results (328)

Bone remodeling relies on a balance between osteoclast-mediated resorption and osteoblast-mediated formation. Roxadustat, a hypoxia-inducible factor prolyl hydroxylase inhibitor, promotes osteoblast differentiation but its effects on osteoclasts remain unclear. This study investigated roxadustat’s impact on osteoclast differentiation and function in vitro using primary murine bone marrow-derived mononuclear cells differentiated with M-CSF and RANKL. Cell viability, TRAP staining, bone resorption assays, RNA-seq, flow cytometry, immunofluorescence, Western blot for p27, and rescue experiments with the cyclin-dependent kinases 4 and 6 (CDK4/6) inhibitor abemaciclib were performed. Roxadustat suppressed osteoclast differentiation and resorption without cytotoxicity in a concentration-dependent manner. RNA-seq revealed enrichment of cell cycle pathways; although differentiation was inhibited, roxadustat paradoxically promoted osteoclast precursor proliferation, evidenced by increased Ki67 and decreased p27 expression. The inhibitory effects on osteoclastogenesis and resorption were partially reversed by abemaciclib. Given that terminal differentiation typically requires cell cycle exit, these findings suggest that roxadustat may inhibit osteoclast differentiation at least in part by disrupting this process, promoting precursor proliferation, and downregulating p27. Together with its known anabolic effects on osteoblasts, roxadustat might have dual therapeutic potential for bone disorders with renal anemia, such as osteoporosis in chronic kidney disease. Full article

Neutrophils are central executors of innate immunity. Yet murine models are inherently limited by low baseline neutrophil counts. NSG mice are among the most widely used models for xenotransplantation and studies on the humanized immune system. Although G-CSF is known to stimulate granulopoiesis, the dose- and schedule-dependent effects of intraperitoneal G-CSF administration have not been systematically characterized in this immunodeficient background. Male NSG mice received intraperitoneal G-CSF according to one of five regimens (n = 6 per group): group 0 served as the saline control, group 1 received a single dose of 250 µg/kg G-CSF administered at 48 h; group 2 received a single dose of 250 µg/kg G-CSF administered at 24 h; group 3 received three doses of 250 µg/kg administered G-CSF at 0 h, 24 h, and 48 h and group 4 received a single dose of 500 µg/kg G-CSF administered at 48 h. All animals were sacrificed at 72 h. Circulating neutrophils were then quantified by flow cytometry, bone marrow neutrophil proportions by panoptic smear analysis, and splenic neutrophil abundance by Ly6G immunofluorescence. Systemic neutrophil activation was assessed via plasma neutrophil elastase (NE) activity and cell-free DNA (cfDNA) levels. Repeated G-CSF administration (Group 3) induced an approximately 13-fold expansion of circulating neutrophils, approaching the human physiological range, with significant increases also observed in bone marrow and a trend towards increased neutrophil abundance in the spleen. A single dose of 250 µg/kg administered at 24 h (group 2), produced significant neutrophil expansion in peripheral blood and bone marrow but not in the spleen, while all other single-dose regimens failed to induce significant expansion in any compartment. NE activity and cfDNA concentrations and a selected cytokine panel remained unaltered across all groups. This systematic comparison establishes repeated intraperitoneal G-CSF administration as a reproducible strategy to achieve human-like neutrophil levels in NSG mice without inducing systemic inflammation. This provides a validated protocol with direct utility in translational models of neutrophil-dependent diseases. Full article

Bone marrow-derived mesenchymal stem cells (BMSCs) maintain skeletal homeostasis by balancing adipogenic and osteogenic differentiation, yet clinically used drugs that bias this fate choice and their mechanisms remain incompletely defined. Here, we investigated whether dextromethorphan (DXM), a widely used antitussive, modulated lineage commitment in rat BMSCs and interrogated candidate upstream signaling modules. Rat BMSCs were induced with adipogenic medium or osteogenic medium in the presence of DXM (30 μM). Adipogenesis and osteogenesis were quantified using Oil Red O and Alizarin Red S staining with elution-based quantification, and lineage markers were measured by RT-qPCR. Intracellular Ca²⁺ and ROS were analyzed using flow cytometry, and the levels of p-AKT and p-ERK were assessed through Western blotting analysis. Under adipogenic induction, DXM increased lipid droplet accumulation and the mRNA levels of Pparγ and Fabp4. Although DXM elevated Ca²⁺ and ROS, the chelation of intracellular Ca²⁺ and pharmacological inhibition of Sig-1R/PLC–IP3R signaling, redox/ROS, NMDA receptors, AKT/ERK, Kv channels, bitter taste receptor-related signaling, and mTOR did not attenuate the DXM-enhanced adipogenesis. DXM reduced p-ERK without increasing p-AKT; U0126 lowered basal adipogenesis but did not block the DXM effect. Under osteogenic induction, DXM reduced matrix mineralization and downregulated Runx2 and Bglap mRNA levels, while Wwtr1 mRNA levels were not significantly changed. DXM also partially reversed the osteogenic induction-associated reduction in Mtor mRNA. Separately, under adipogenic induction, rapamycin attenuated baseline adipogenesis but did not prevent the additional lipid accumulation induced by DXM. Collectively, DXM shifted the osteogenic–adipogenic balance toward adipogenesis through a non-canonical mechanism. Full article

Multiple myeloma (MM) develops through asymptomatic precursor stages characterized by progressive remodeling of the bone marrow (BM) immune microenvironment and disruption of bone homeostasis. To delineate changes in natural killer (NK) cell states during disease evolution, we investigated coordinated immune-tumor remodeling by integrating NK cell functional states with plasma cell-intrinsic susceptibility programs derived from CRISPR-based screens across healthy donors (HD), monoclonal gammopathy of undetermined significance (MGUS), smoldering MM (SMM), and newly diagnosed MM patients. The integration of NK cell state-associated gene signatures with plasma cell transcriptional programs revealed stage-specific co-variation between immune and tumor compartments. Public single-cell RNA sequencing datasets were interrogated to resolve NK cell heterogeneity, identifying cytotoxic CD56^dim and regulatory CD56^bright subsets. NK cell dynamics displayed stage-dependent changes, with early expansion followed by the contraction of CD56^dim cells in BM, whereas CD56^bright cells showed predominantly compositional remodeling. Within the CD56^bright subset, transcriptional changes included an increased expression of KLRC1 (encoding NKG2A), subsequently validated by multiparametric flow cytometry. In parallel, plasma cell programs associated with NK sensitivity progressively decreased along disease stages, supporting tumor adaptation to immune pressure. The NKG2A ligand HLA-E displayed selective expression within CD16⁺ monocytes and followed a distinct variable pattern across disease stages, highlighting a microenvironmental contribution to NK cell regulation. Collectively, these findings indicate a coordinated process of immune-tumor co-evolution, characterized by dynamic remodeling of NK cell states and plasma cell susceptibility, with the NKG2A–HLA-E axis emerging as a central interface during MM progression. Full article

Background: Colorectal cancer is a common malignancy and 5-fluorouracil (FU) remains a mainstay of chemotherapy despite its toxicity. As an important part of comprehensive tumor treatment, dual-protein (DP) nutritional intervention is attracting more and more attention. Methods: This study preliminarily evaluated the regulatory effects of DP intervention on colorectal cells of CT26 tumor-bearing mice, examining the dosage and administration methods of DP, as well as the anti-tumor effects of FU alone or in combination with DP. Results: The results showed that low- and medium-dose DP numerically increased spleen index and showed trends toward alleviating FU-induced thymic atrophy, splenic damage, nephrotoxicity, and myocardial injury. It also partly mitigated muscle wasting, prevented FU-induced shortening of the colorectal tract, and reduced intestinal injury. In addition, DP was associated with increased lymphocyte, monocyte, and platelet counts and decreased granulocytes, suggesting possible alleviation of chemotherapy-induced bone marrow suppression and a potential effect on hematopoietic function. Flow cytometry results indicated possible effects of DP on CD4⁺ T and CD8⁺ T cell proliferation or apoptosis, modulation of effector and memory phenotypes, reduced splenic neutrophil levels, balanced B cell function, and maintained natural killer cell activity. In addition, DP intervention also showed trends toward regulating hepatic lipid metabolism and partially alleviating FU-induced dyslipidemia and muscle damage. In addition, DP and FU could increase IL-2, IL-10, GM-CSF and IFN-γ and decrease IL-6 and TNF-α. Conclusions: In conclusion, a moderate dose (0.67 g/kg) of DP had the most favorable trends, and the pre-intervention mode was more effective. This study also provided exploratory data on the potential of DP in reducing chemotherapy-related toxicity. These findings will provide preliminary scientific support for nutritional therapy in colorectal cancer patients, as well as for the research, development, and application of dual-protein foods for special medical purposes. Full article

Antigen-presenting cells (APCs) play a critical role in modulating immune responses, making the optimization of their differentiation protocols essential for advancing cell-based immunotherapies. This study evaluated eight protocols to differentiate APCs from bone marrow precursors of C57BL/6J mice, comparing the effects of GM-CSF and L-929 conditioned supernatants at various concentrations. Four groups treated with GM-CSF and four with L-929 supernatant, alongside a control group, were assessed. Flow cytometry analysis revealed that GM-CSF significantly increased the yield of CD11c⁺ MHC II⁺ cells by up to 6-fold compared to the L-929 supernatant. Furthermore, GM-CSF-treated groups showed higher mean fluorescence intensities (MFI) for critical markers such as MHC II and CD11c, with MFI levels surpassing those of SL-929-treated groups by approximately 3- to 5-fold. In contrast, the L-929 supernatant demonstrated limited efficacy in promoting both cell differentiation and surface marker expression, resulting in minimal phenotypic and quantitative gains compared to controls. These findings highlight the superior efficiency of GM-CSF in driving APC differentiation and underscore the importance of balancing cell yield and phenotypic fidelity when selecting differentiation protocols. This study provides valuable insights for researchers developing targeted immunotherapies and offers a solid foundation for optimizing APC-dependent therapies, ensuring efficacy and cost-efficiency in cell-based strategies. Full article

Natural killer T (NKT) cells bridge innate and adaptive immune responses and play a critical role in anti-tumor immunity. The goal of the study was to assess NKT cell and T cell function in lymphoma patients and to investigate whether specific cytokines correlate with outcomes and/or immune cell function. Patient diagnoses were confirmed by histology. NKT and T cell number and function were assessed by flow cytometry and stimulation with artificial antigen-presenting cells (aAPCs) followed by ELISA and quantitative RT-PCR (qPCR). Cytokine expression levels were compared using online databases, and protein levels in the plasma were assessed by ELISA. NKT cell activation, indicated by at least 1.5-fold IFN-γ induction over baseline following stimulation, was detected in 82% of healthy donors, compared to 44% of lymphoma patients. Lymphoma patients have significantly higher levels of circulating pro- and anti-inflammatory cytokines IL-10, IL-6, and Sema4D as compared to healthy donors. In addition, NKT cell function in the blood correlated with NKT cell function in the bone marrow in lymphoma patients. We found that aAPC-qPCR can be used to quickly assess immune cell function in cancer patients. Circulating NKT cell function positively correlated with bone marrow NKT cell function, suggesting that circulating NKT responses reflect systemic immune competence. Outcome-associated transcriptomic analyses showed that lower expression of TGF-β, IL-6, IL-10, and IFN-γ mRNA correlated with poorer clinical outcomes, whereas higher Sema4D expression was associated with worse prognosis, identifying Sema4D as a potential immunologic biomarker linked to disease progression and immune dysfunction in B cell lymphoma. Full article

Background and Aims: Anemia is seen in nearly >70% of patients with cirrhosis and is often non-responsive to nutritional supplements; therefore, we assessed the erythropoiesis and associated alteration in bone marrow (BM). Methods: It is a cross-sectional study. Flow cytometry was performed to assess the hematopoietic stem cells (HSCs) and erythroid population of 60 patients with cirrhosis compared with patients with 7 non-cirrhotic portal fibrosis (NCPF) and 3 controls. Proteomics were performed of the pure CD71 erythroid population taken from patients with cirrhosis to decipher the internal abnormalities supported by validation experiments. Real Time PCR, colony assay and heme quantification, cytokine array, and ELISA were performed to assess erythropoietic stimulating agents (ESA), inflammatory cytokines, and growth factors as an external factor affecting erythropoiesis. Results: We found a decrease in intermediate erythroid progenitors [IEPs; CD71+ CD235a+], conversely early erythroid precursors [EEP; CD71+ CD235a−] and late erythroid progenitors [LEP; CD71− CD235a+] were increased (p < 0.05) in cirrhotic and NCPF as compared to control. However, unlike NCPF, cirrhosis exhibited decreased CD71+ transferrin receptor (TfR1) expression over erythroid cells and increased immature erythrocytes (p < 0.05) in peripheral circulation. In vitro culture of erythroid precursors showed impaired differentiation and maturation that was confirmed by the reduced (p < 0.05) number of erythroid colonies (BFU-E). Proteomics analysis showed downregulated proteins associated with hemoglobin synthesis, ROS detoxification, translation, and mitochondrial activity. Furthermore, we found an altered expression of genes related to erythropoiesis and hemoglobin synthesis and increase (p < 0.05) in inflammatory cytokines such as IL-5, TRAIL-R2, TGF-α, and TGF-β in BM. Conclusions: This study suggests that the dysregulated erythropoiesis observed in patients with cirrhosis having anemia is maintained despite adequate nutrition. Full article

Osteoarthritis (OA) is a degenerative, age-related joint disease involving bone remodeling and damage to articular cartilage. OA is the most common form of arthritis, causing pain, swelling, stiffness, and reduced mobility. Given the limited efficacy of current therapies, there is growing interest in natural compounds with anti-inflammatory and immunomodulatory properties. Crocus sativus L., known as saffron, contains more than 150 biologically active compounds with proven antioxidant and anti-inflammatory effects, making it a promising candidate for modulating OA-related processes. The aim of the study was to evaluate the effects of saffron extract on immune cell function, osteoclast differentiation, and joint pathology in collagenase-induced osteoarthritis (CIOA) mouse model. OA was induced by intra-articular injection of Collagenase type IA, followed by daily treatment with saffron extract for 30 days. Flow cytometry, apoptosis, Western blot and proliferation assays were performed to analyze the phenotype and activity of bone marrow and synovium cells, as well as histological evaluation of joint tissues. Saffron therapy promoted an anti-inflammatory immune profile, reduced T-cell apoptosis and proliferation, and inhibited osteoclast differentiation. These changes were accompanied by improved histological manifestations of the joints. Overall, the findings suggest that saffron may modulate key inflammatory and cellular mechanisms involved in OA, although further research is needed to confirm its therapeutic relevance in humans. Full article

Extracellular vesicles (EVs) are lipid bilayer-bound structures capable of transporting molecular markers from their cell of origin and are secreted by multiple cell types, including malignant cells. EVs have emerged as promising tools for developing less invasive diagnostic approaches. In B-cell acute lymphoblastic leukemia (B-ALL), immunophenotypic characterization of extracellular vesicle-enriched populations (EVEPs) in peripheral blood (PB) may provide complementary information for disease detection and monitoring. This exploratory study aimed to characterize EVEPs obtained from PB and bone marrow (BM) of adult patients with B-ALL and to compare them with the clinical immunophenotype (CIP). A total of 12 individuals were initially recruited (eight with B-ALL, one with T-ALL, and three healthy controls). The study focused on the eight B-ALL patients and three controls, while the T-ALL sample was used as a specificity control. EVEPs were isolated by differential centrifugation and analyzed by flow cytometry and confocal microscopy, primarily evaluating CD3 and CD19 expression. EVEPs derived from PB samples of patients with B-ALL showed a higher percentage of marker-positive events by flow cytometry (CD45, CD34, CD19, CD20, and CD10), consistent with the leukemic phenotype identified in the CIP. Additionally, CD3⁺CD19⁺ EVEPs were occasionally detected. These findings suggest that EVEPs partially reflect the leukemic immunophenotype and may serve as a complementary source of biological information. The detection of CD3⁺CD19⁺ events highlights complex cellular interactions within the leukemic niche and warrants further investigation. Full article

Mesenchymal stromal cells (MSCs) have shown immunomodulatory effects and great promise in many inflammatory diseases such as acute respiratory distress syndrome (ARDS). However, several barriers to translation remain such as cell availability and potency. This study evaluates the therapeutic potentials of three types of MSCs, bone marrow-derived MSCs (BM-MSC), the human induced pluripotent stem cell-derived MSC wild type (iMSC WT) and β2 microglobulin-knockout iMSCs (iMSC B2M KO) with or without proinflammatory cytokine preconditioning. BM-MSC, iMSC WT and iMSC B2M KO were preconditioned with a proinflammatory cytokine cocktail (Cytomix: IL-1β, IFN-γ and TNF-α). Immunoregulatory biomarkers were analysed by flow cytometry and cytokines released by ELISA. MSC antimicrobial properties were analysed via CFU assays while the MSCs’ immunomodulatory effects were evaluated using macrophage activation and T cell proliferation assays. Proinflammatory cytokine preconditioning enhanced the therapeutic potency of all three types of MSCs by increasing immunomodulatory marker expression, enhancing the antimicrobial effects and improving MSC-mediated inhibition of T cell proliferation. These findings provided new insights into the therapeutic potencies of MSCs in inflammation. Further studies are required for in vitro characterisation of the MSCs and in vivo efficacy verification of these MSCs prior to their clinical application. Full article

Background/Objectives: Pediatric cancers are disorders of dysregulated development driven largely by genomic and epigenetic alterations. Precisely modeling these developmental differences is essential for understanding the unique biology of childhood cancers. Patient-derived organoids (PDOs) offer a powerful in vitro platform that recapitulates tumor heterogeneity, plasticity, microenvironment (including immune cells) and disease-relevant features. Methods: Here, we describe a step-by-step protocol for the establishment of PDOs from cells derived from pediatric brain tumors and extracranial solid tumor biopsies and bone marrow aspirates, including tumor processing, organoid culture/subculture, and cryopreservation. Results: Furthermore, we present the use of PDOs for further experimental analysis such as fluorescence imaging, Western blotting, flow cytometry, and immunohistochemistry (IHC) to investigate the underlying pathophysiology of tumorigenesis. Conclusions: Expanding the application of organoids to childhood malignancies holds exceptional promise for elucidating pediatric tumor biology and advancing therapeutic strategies, representing the long-overdue convergence of technology and clinical need. Full article

The immunological composition of the microenvironment has shown relevance for diagnosis, prognosis, and therapy in solid tumors but remains underexplored in acute leukemias. We investigated the significance of the acute myeloid leukemia (AML) bone marrow microenvironment in predicting chemosensitivity and long-term remission in pediatric patients. We analyzed 32 non-promyelocytic pediatric AML patients at diagnosis using a NanoString PanCancer IO 360 assay, RNA sequencing, and deep-phenotype flow cytometry analyses. The findings were validated using the pediatric TARGET AML dataset. A short signature of three interferon (IFN)-related genes (GBP1, PARP12, and TRAT1) distinguished patients with chemosensitive disease and reduced minimal residual disease after induction chemotherapy. The signature stratified patients overall, and within the clinically defined “standard-risk” group, patients with high gene expression at diagnosis had significantly longer overall survival. The leukemia microenvironment associated with this signature showed enrichment of non-exhausted CD4⁺ and CD8⁺ T cytotoxic lymphocytes and expansion of CD8⁺ T effector memory cells re-expressing CD45RA (TEMRA) in patients with a favorable prognosis. Our results show the importance of the bone marrow microenvironment in pediatric AML and provide tools for a refined stratification of “standard-risk” patients, lacking adequate risk-oriented therapies. They also offer a promising guide for tackling immune pathways and exploiting immune-targeted therapies. 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

Background/Objectives: Serum protein electrophoresis (SPE) is a widely used laboratory test for the detection and monitoring of monoclonal gammopathies, including multiple myeloma (MM). Although SPE is usually recommended in the presence of specific clinical or laboratory abnormalities, monoclonal gammopathies may occasionally develop rapidly and without typical symptoms. This case report aims to emphasize the diagnostic value of SPE in identifying an unexpected and fast-evolving monoclonal gammopathy. Methods: We report the clinical and laboratory eight-month follow-up of a 58-year-old male who initially underwent SPE for unrelated clinical conditions. Serial SPE analyses were performed using capillary zone electrophoresis. When abnormalities emerged, immunotyping and serum free light chain (FLC) assays were conducted. The diagnostic workup was completed with bone marrow aspiration, flow cytometry, and imaging studies according to current international diagnostic criteria. Results: The initial SPE (November 2023) showed a normal protein profile. After eight months, follow-up SPE revealed a prominent monoclonal spike in the gamma region (2.9 g/dL), associated with increased total serum proteins (91 g/L; range 64–82 g/L), elevated IgA levels (20.0 g/L; range 0.4–3.5 g/L), and a markedly abnormal κ/λ FLC ratio (54.00; range 0.31–1.56). Bone marrow analysis demonstrated >18% plasma cell infiltration, confirming the diagnosis of IgA-κ MM. The patient underwent standard therapy followed by autologous stem cell transplantation, achieving disease remission. Conclusions: This case highlights that clinically relevant monoclonal gammopathies may arise rapidly in the absence of classical diagnostic features. Routine SPE represents a cost-effective and accessible screening tool that can identify subtle protein abnormalities, prompting the timely use of more specific and invasive diagnostic procedures for aggressive plasma cell disorders. Full article