Profile Of Cells Research Articles

Associations between parity, history of breastfeeding, and T cell profile of ovarian tumors.

Parity and breastfeeding are associated with systemic changes in maternal inflammation and reduced risk of ovarian cancer, but little is known about their impact on the ovarian tumor immune microenvironment. We evaluated the associations of self-reported parity and history of breastfeeding with tumor-infiltrating T cells among 1,706 ovarian carcinoma cases with tumor tissue collected across four studies. The abundance of tumor-infiltrating T cells was measured by multiplex immunofluorescence in tumor tissue microarrays. Odds ratios (OR) and 95% confidence intervals (CIs) for the positivity of tumor immune cells were calculated using beta-binomial models and stratified by histotype. Compared to ovarian tumors in nulliparous women, there was no association between parity and ovarian tumor T cell abundance among all histotypes combined, but suggestion of increased cytotoxic T cells and T cell exhaustion among parous women with clear cell tumors. When restricted to parous women, history of breastfeeding was associated with increased odds for all T cell types (i.e., total, cytotoxic, helper, regulatory, and exhausted T cells), with ORs ranging from 1.11-1.42. For every 6 months of breastfeeding, we observed increased odds of activated helper T cell infiltration (CD3+CD4+CD69+, OR:1.13, 95% CI: 0.99-1.29), with a similar association for high-grade serous tumors, but lower odds in clear cell tumors (OR:0.43, 95% CI:0.21-0.87). History of breastfeeding may alter the ovarian tumor immune microenvironment by modulating the abundance of tumor-infiltrating T cells. While replication is required, history of breastfeeding may play a role in activation of the ovarian tumor immune response.

Cryptogenic cirrhosis and Common variable immunodeficiency: an unrecognized relationship

Objective: This study investigates the prevalence of Common Variable Immunodeficiency (CVID) in patients with cryptogenic cirrhosis. It aims to highlight CVID's role in liver involvement and raise awareness of its potential as an underlying cause of cryptogenic cirrhosis. Methods: This retrospective cohort study, approved by the ethics committee, included patients diagnosed with cryptogenic cirrhosis at a university hospital. Comprehensive evaluations were performed to exclude other causes of cirrhosis, and patients were screened for CVID based on the European Society for Immunodeficiencies (ESID) criteria. Results: This study included 30 patients with cryptogenic cirrhosis, among whom 6.7% (n=2) were diagnosed with CVID. Both CVID patients had low immunoglobulin levels and abnormal immune cell profiles, leading to recurrent infections in one case and suspicion due to low total protein levels in the other. IVIG treatment was initiated for both, and liver biopsy findings in one patient suggested CVID-related liver involvement. Conclusions: Determining the underlying causes of chronic liver diseases is crucial for guiding treatment and follow-up, potentially preventing cirrhosis progression and influencing liver transplantation eligibility. CVID screening in patients with cryptogenic cirrhosis should be considered, with appropriate treatments initiated as needed.

Cyanidin-3-glucoside reduces cell migration and inflammatory profile of acute leukemia cells.

Anthocyanins, water-soluble flavonoids found in fruits and vegetables, exhibit diverse biological activities, with cyanidin being the most common pigment. While cyanidin's chemo preventive and -antioxidant activity is well-studied, its impact on inflammatory profile and migration capacity of leukemic cells are less understood. This study evaluates the effects of Cyanidin-3-glucoside (C3G) on the inflammatory mechanisms influencing leukemic cell migration. Results show that C3G doses up to 50 µM do not affect cell metabolism, viability, or cell cycle phases. C3G significantly reduces TNF-α, IL-8, CCL2 production, and the p-NFκB/NFκB ratio in LPS (Lipopolysaccharide)-challenged cells. It also diminishes migration rates in response to LPS or fMLP (N-formyl-methionyl-leucyl-phenylalanine) stimulation and reduces Rho-GTP expression. Thus, C3G modifies the inflammatory and migration properties of leukemic cells, highlighting the potential of anthocyanins as a complementary therapy and an avenue for further therapeutic intervention.

Single cell RNA sequencing of hematopoietic cells in fresh and frozen human atheroma tissue.

Single-cell RNA sequencing (scRNA-seq) is a powerful method for exploring the cellular heterogeneity within human atheroma but typically requires fresh tissue to preserve cell membrane integrity, limiting the feasibility of large-scale biobanking for later analysis. The aim of this study was to determine whether cryopreservation of fragile and necrotic atheroma tissue affects the viability and transcriptomic profiles of hematopoietic cells in subsequent scRNA-seq analysis, enabling the use of cryopreserved atheroma samples for future research. We performed scRNA-seq on five paired fresh and cryopreserved atheroma samples - three from coronary arteries and two from carotid arteries. Each sample was enzymatically digested, sorted for CD45+ hematopoietic cells, and processed using the 10X Genomics scRNA-seq workflow. Half of each sample was processed immediately, while the other half was cryopreserved in liquid nitrogen for an average of five weeks before thawing and processing. In carotid artery samples, we noted the absence of LYVE1+ macrophages, likely due to the loss of the adventitial layer during endarterectomy procedures. Our results indicated that cryopreservation modestly affected cellular integrity, leading to an increase in the relative abundance of mitochondrial RNA in frozen samples. Minimal differences were observed between fresh and cryopreserved samples in uniquely detected transcripts, cell clustering, or transcriptional profiles within hematopoietic populations. Our study demonstrates that cryopreserved human atheroma samples can be successfully profiled using scRNA-seq, with comparable transcriptomic data to that obtained from fresh samples. These findings suggest that cryopreservation is a viable method for biobanking atheroma tissues, facilitating large-scale studies without the need for immediate sample processing.

Development and evaluation of a novel E7 multi-epitopic vaccine for human papillomavirus type 16: design, expression, purification, and immunological characterization

BackgroundPersistent infection with high-risk Human papillomavirus (HPV), specifically HPV-16, is the leading cause of cervical cancer. Although preventative vaccines have shown significant efficacy in preventing HPV infection, cervical cancer is a significant public health issue that affects millions of women worldwide. Modern therapeutic approaches, such as peptide vaccines, could be promising and have potential for the treatment of the HPV-infected population.MethodsA HPV16-E7 multi-epitopic vaccine (MEVE7) was designed to comprise potent CD4 + and CD8 + T cell epitopes and optimally expressed in a prokaryotic expression system. Polyclonal antibodies were generated, and their reactivity with immunizing antigen and native protein in E7 expressing cells (TC-1) was assessed by ELISA and immunofluorescent staining, respectively. The efficacy of the vaccine was assessed in a therapeutic animal model of HPV-induced cancer.ResultsOur study revealed that the final construct was successfully expressed in E. coli BL21 (DE3)-gold within 4 h of induction as inclusion bodies. Among the tested solubilization buffers, the buffer with a pH of 12 and containing 2 M urea showed the highest solubilization effect. Polyclonal antibodies directed against the E7 multi-epitope vaccine were able to react strongly with the immunizing antigen and E7-bearing cells (TC-1). Immunization of TC-1 tumor-bearing mice with HPV16-E7, markedly delayed tumor growth and propagation.ConclusionThe poly-epitope vaccine for HPV16-E7, as expressed and purified in this research, is highly immunogenic and capable of triggering E7-specific antibodies, making it a potential therapeutic HPV vaccine. Further research is needed to optimize the vaccination schedule and assess the E7-specific immune cell profile.

Patient-Derived Cancer-Associated Fibroblasts Support the Colonization of Tumor Cells in Head and Neck Squamous Cell Carcinoma

Background: The crosstalk between cancer-associated fibroblasts (CAFs) and tumor cells promotes proliferation, tumor relapse, and the acquisition of a partial epithelial-to-mesenchymal (pEMT) phenotype in tumor cells. The aim of this study was to investigate the effects of patient-derived CAFs on tumor cell growth and radioresistance in head and neck squamous cell carcinoma (HNSCC). Methods: CAFs were isolated and cultured in a three-dimensional spheroid formation. SCC-25 tumor cells educated by the CAFs (SCC25-E cells) were subjected to irradiation, and the response of the CAF-stimulated tumor cells to radiotherapy was determined using an MTT assay, a clonogenic assay, and Western blotting. Tumor cell morphological changes and growth dynamics were assessed using 3D holotomographic microscopy and a live video microscope. Results: Patient-derived CAFs significantly increased the growth rate of SCC-25 cells. CAFs drove fibrosis in the tumor microenvironment (TME), functioned as a physical barrier, temporarily stopped tumor growth, and induced the p38 mitogen-activated protein kinase (MAPK) signaling pathway. Viability after irradiation at 4–8 Gy was significantly higher in SCC25-E cells than in the controls (p = 8 × 10–4 or lower). Furthermore, irradiation triggered the pEMT profile in HNSCC cells. Conclusions: CAFs’ education of tumor cells and the induced p38 phosphorylation had no influence on irradiation sensitivity. SCC25-E cultures demonstrated increased tumor cell growth, viability, and stress-induced phospho-p38 activation.

Immune cell profiles and predictive modeling in osteoporotic vertebral fractures using XGBoost machine learning algorithms

BackgroundOsteoporosis significantly increases the risk of vertebral fractures, particularly among postmenopausal women, decreasing their quality of life. These fractures, often undiagnosed, can lead to severe health consequences and are influenced by bone mineral density and abnormal loads. Management strategies range from non-surgical interventions to surgical treatments. Moreover, the interaction between immune cells and bone cells plays a crucial role in bone repair processes, highlighting the importance of osteoimmunology in understanding and treating bone pathologies.MethodsThis study aims to investigate the xCell signature-based immune cell profiles in osteoporotic patients with and without vertebral fractures, utilizing advanced predictive modeling through the XGBoost algorithm.ResultsOur findings reveal an increased presence of CD4 + naïve T cells and central memory T cells in VF patients, indicating distinct adaptive immune responses. The XGBoost model identified Th1 cells, CD4 memory T cells, and hematopoietic stem cells as key predictors of VF. Notably, VF patients exhibited a reduction in Th1 cells and an enrichment of Th17 cells, which promote osteoclastogenesis and bone resorption. Gene expression analysis further highlighted an upregulation of osteoclast-related genes and a downregulation of osteoblast-related genes in VF patients, emphasizing the disrupted balance between bone formation and resorption. These findings underscore the critical role of immune cells in the pathogenesis of osteoporotic fractures and highlight the potential of XGBoost in identifying key biomarkers and therapeutic targets for mitigating fracture risk in osteoporotic patients.

Autoimmunity promotes chronic lymphocytic leukemia progression in an indolent disease model

Chronic lymphocytic leukemia (CLL) is a heterogeneous B cell malignancy characterized by the accumulation of functionally incompetent B lymphocytes. Despite the availability of highly effective treatments, CLL remains incurable, and the factors contributing to disease progression are not fully understood. Autoimmune complications frequently arise in CLL patients and are associated with poor clinical prognosis. This study investigates the connection between plasma cell-mediated autoimmunity and CLL progression using a mouse model that expresses an active Receptor Activator of NF-κB (RANK) in B cells (RK mice), where autoimmune manifestations coexist with CLL. Transcriptional profiling of RANK-driven leukemic cells revealed a more indolent form of CLL compared to the classical TCL1 model. The discovery of near-identical CDR3 regions in both plasma and CLL cells of RK mice suggests a shared progenitor and antigen driving both conditions. Deletion of Blimp-1, which prevents plasma cell differentiation, initially enhanced B1/CLL formation in young mice but nearly halted CLL progression, highlighting the significant influence of autoimmune complications on disease outcomes. This research underscores the intertwined nature of autoimmunity and CLL, suggesting that targeting inflammatory pathways could offer therapeutic potential for managing both conditions.

Prognostic Value of Ki-67 in the Invasive Zone of Oral Squamous Cell Carcinoma.

The molecular profile of cells within the tumor invasive zone, where tumor cells interact with surrounding non-tumor cells, plays a crucial role in defining tumor malignant characteristics, such as the pattern of invasion (POI). Therefore, evaluating the diagnostic value of the proliferation index molecule, Ki-67, in both tumor cells and adjacent non-tumor cells at the invasive zone with different POIs, holds significant clinical importance for oral squamous cell carcinoma (OSCC). This retrospective study included 133 primary OSCC samples, and the spatial pattern of Ki-67 in the tumor invasive zone was analyzed by immunohistochemistry (IHC). The prognostic value of tumor cells and stroma proliferative capacity in different POIs were assessed. Ki-67 was widely expressed in tumor cells and stroma cells within the invasive zone, and cells in high-invasiveness POIs exhibit higher proliferation. Elevated Ki-67 expression in tumor cells was associated with poor overall survival (OS) and disease-free survival (DFS) in patients, which is independent of POIs. In our study, we identified the expression level of Ki-67 in tumor cells across high-invasiveness POIs as an independent risk factor for OS and DFS in OSCC patiens. Additionally, Ki-67 expression in surrounding non-tumor cells did not significantly correlate with patient survival. The remarkable proliferation characteristic of tumor cells in high-invasiveness POIs of OSCC tumors plays a crucial role in the prognosis of patients.

Unraveling the tumor microenvironment of esophageal squamous cell carcinoma through single-cell sequencing: A comprehensive review.

Esophageal squamous cell carcinoma (ESCC) is a highly heterogeneous and aggressive malignancy. The progression, invasiveness, and metastatic potential of ESCC are shaped by a multitude of cells within the tumor microenvironment (TME), including tumor cells, immune cells, endothelial cells, as well as fibroblasts and other cell types. Recent advancements in single-cell sequencing technologies have significantly enhanced our comprehension of the diverse landscape of ESCC. Single-cell multi-omics technology, particularly single-cell transcriptome sequencing, have shed light on the expression profiles of individual cells and the molecular characteristics of distinct tumor cell populations. This review summarizes the latest literature on single-cell research in the field of ESCC, aiming to elucidate the heterogeneity of tumor cells, immune cells, and stromal cells at the single-cell level. Furthermore, it explores the impact of cellular interactions within the TME on the progression of ESCC. By compiling a comprehensive overview of single-cell omics research on ESCC, this article aims to enhance our understanding of ESCC diagnosis and treatment by elucidating the intricate interplay within the TME. It explores the cellular composition, spatial arrangement, and functional attributes of the ESCC TME, offering potential therapeutic targets and biomarkers for personalized treatment strategies.

The COVID-19 vaccine ChAdOx1 is opsonized by anti-vector antibodies that activate complement and promote viral vector phagocytosis.

The ChAdOx1 nCoV-19 vaccine has been in large-scale use during the COVID-19 pandemic. Limited efficacy compared to mRNA vaccines and certain potential side effects raise the question of whether anti-adenoviral vector antibodies influence immune responses against the vaccine. Complement activation by ChAdOx1 and leukocyte phagocytosis of ChAdOx1 invitro were studied. Plasma IgG levels against ChAdOx1 and human adenovirus 2 (hAdV2) hexon protein were determined (n = 20) and IgGs from high- and low-titre plasmas were isolated (n = 3). Complement activation was measured as cleavage of C3 by immunoblotting and generation of C3a and sC5b-9 by ELISA. pHrodo-labelled ChAdOx1 was opsonized with complement and IgG, and phagocytosis by isolated blood PMNs invitro was studied by flow cytometry. The transcriptomic profile of PMN cells exposed to ChAdOx1 was analysed by RNA-seq. ChAdOx1 activated the classical complement pathway in an anti-adenovirus antibody-dependent manner. Generation of the terminal complement complex sC5b-9 in individual sera correlated with anti-hAdV2 hexon and anti-ChAdOx1 IgG levels. Phagocytosis of ChAdOx1 also correlated significantly with anti-hAdV2 hexon IgG, anti-ChAdOx1 IgG and serum sC5b-9 levels. High-titre anti-hAdv2 hexon IgG increased phagocytosis in the presence of normal serum. Anti-vector antibodies induced rapid complement activation and promoted phagocytosis of the ChAdOx1 vaccine by neutrophils. Moreover, transcriptomic analysis revealed upregulation of complement-related genes induced by the ChAdOx1 vaccine invitro. Anti-adenovirus vector antibodies and complement activation may thus influence the efficacy of the ChAdOx1 vaccine against SARS-CoV-2 and be also involved in vaccine-related side effects.

Sonic hedgehog medulloblastoma cells in co-culture with cerebellar organoids converge towards in vivo malignant cell states.

In the malignant brain tumor sonic hedgehog medulloblastoma (SHH-MB) the properties of cancer cells are influenced by their microenvironment, but the nature of those effects and the phenotypic consequences for the tumor are poorly understood. The aim of this study was to identify the phenotypic properties of SHH-MB cells that were driven by the nonmalignant tumor microenvironment. Human induced pluripotent cells (iPSC) were differentiated to cerebellar organoids to simulate the nonmaliganant tumor microenvironment. Tumor spheroids were generated from 2 distinct, long-established SHH-MB cell lines which were co-cultured with cerebellar organoids. We profiled the cellular transcriptomes of malignant and nonmalignant cells by performing droplet-based single-cell RNA sequencing (scRNA-seq). The transcriptional profiles of tumor cells in co-culture were compared with those of malignant cell monocultures and with public SHH-MB datasets of patient tumors and patient-derived orthotopic xenograft (PDX) mouse models. SHH-MB cell lines in organoid co-culture adopted patient tumor-associated phenotypes and showed increased heterogeneity compared to monocultures. Subpopulations of co-cultured SHH-MB cells activated a key marker of differentiating granule cells, NEUROD1 that was not observed in tumor monocultures. Other subpopulations expressed transcriptional determinants consistent with a cancer stem cell-like state that resembled cell states identified in vivo. For SHH-MB cell lines in co-culture, there was a convergence of malignant cell states towards patterns of heterogeneity in patient tumors and PDX models implying these states were non-cell autonomously induced by the microenvironment. Therefore, we have generated an advanced, novel in vitro model of SHH-MB with potential translational applications.

Nuclear talin-1 provides a bridge between cell adhesion and gene expression.

Talin-1 (TLN1) is best known to activate integrin receptors and transmit mechanical stimuli to the actin cytoskeleton at focal adhesions. However, the localization of TLN1 is not restricted to focal adhesions. By utilizing both subcellular fractionations and confocal microscopy analyses, we show that TLN1 localizes to the nucleus in several human cell lines, where it is tightly associated with the chromatin. Importantly, small interfering RNA (siRNA)-mediated depletion of endogenous TLN1 triggers extensive changes in the gene expression profile of human breast epithelial cells. To determine the functional impact of nuclear TLN1, we expressed a TLN1 fusion protein containing a nuclear localization signal. Our findings revealed that the accumulation of nuclear TLN1 alters the expression of a subset of genes and impairs the formation of cell-cell clusters. This study introduces an additional perspective on the canonical view of TLN1 subcellular localization and function.

142 Longitudinal immune cell profiling and clinical features in obese lupus patients

142 Longitudinal immune cell profiling and clinical features in obese lupus patients

Adjuvant cytokine-induced killer cell immunotherapy in hepatocellular carcinoma: Extended follow-up of a randomized controlled trial and post-treatment immune cell profiling.

518 Background: Notwithstanding the pressing need for adjuvant therapy for hepatocellular carcinoma (HCC), most adjuvant therapies, including atezolizumab/bevacizumab, have failed. Meanwhile, adjuvant immunotherapy utilizing autologous cytokine-induced killer (CIK) cells for HCC improved recurrence-free survival (RFS) in a previously reported randomized controlled trial (RCT) and real-world data. This study aimed to assess the longer-term outcomes of the RCT and elucidate the underlying mechanisms of sustained effects of CIK cell treatment. Methods: This study comprised two parts: a long-term follow-up of the preceding RCT and an analysis of immune cells in patients who received adjuvant CIK cell treatment. In the original RCT, 226 patients who underwent curative treatment for stage I or II HCC were randomly allocated to either the CIK (n=114, 16 injections of 6.4×10 9 CIK cells over an 11-month period) or control group (n=112). The follow-up period was extended to 9 years after the enrollment of the last patient. The primary endpoint was recurrence-free survival (RFS). The secondary endpoints included cancer-specific survival (CSS) and overall survival (OS). Parallelly, a prospective study was conducted to investigate post-treatment changes of immune cells in peripheral blood of 7 patients, who received repeated transfer of CIK cells after curative treatment for HCC, using flow cytometry. Results: In the extended follow-up of the RCT (median follow-up=115.7 months, interquartile range=74.2–130.5 months), the CIK group sustained a significantly prolonged RFS (median=43.5 vs 27.4 months; hazard ratio [HR]=0.74, 95% confidence interval [CI]=0.55–0.99, P =0.045) and CSS (median=unreached; HR=0.49, 95% CI=0.25–0.95, P=0.04) compared to the control group. Adjuvant CIK cell therapy reduced the risk of overall death by 30%, although it did not achieve statistical significance (median=unreached; HR=0.70, 95% CI=0.44–1.11, P=0.1). A preliminary analysis of peripheral blood immune cells revealed that the CIK cell treatment tended to increase the frequencies of CD8 + and CD4 + classical memory cells. Conclusions: Adjuvant CIK cell treatment demonstrated significantly enhanced RFS and CSS in the prolonged follow-up of the RCT extending to 9 years in patients who received curative treatment for HCC. The CIK group also demonstrated a consistent trend of improved OS. The increase in memory T cell populations might be linked to the sustained off-treatment anti-tumor efficacy of CIK cell treatment.

Supplementation with heat-sterilized Lactobacillus crispatus strain KT-11 stimulates the T cell-related immune function of healthy Japanese adults: A pilot randomized, placebo-controlled, double-blind, parallel-group study.

Viral infections are a global public health threat, reaffirming the importance of immune function. We previously identified Lactobacillus crispatus strain KT-11 (KT-11) and found that heat-sterilized KT-11 affect counts of T cell and dendritic cell in vitro, as well as promoted immunoglobulin A production and prevented weight loss caused by influenza virus infection in vivo. It was hypothesized that heat-sterilized KT-11 affects immune cell count/activity even in healthy individuals. We conducted a pilot study to examine the design to verify the effects of heat-sterilized KT-11 supplementation on immune cell count/activity and physical condition. This was a pilot randomized, double-blind, placebo-controlled, parallel-group study including 22 healthy adults who consumed either KT-11 or placebo for 4 weeks. Immunological status (immune cell count/activity and its score) and various immune-related indicators including SARS-CoV-2 immunoglobulin G antibody were assessed, and a physical condition questionnaire was administered. The primary outcome was immune cell count/activity (T cell subsets, B cell, natural killer cell) and overall immunological status score after 4 weeks. Two patients were excluded because of noncompliance; the final analysis included 20 participants (10 participants/group). The KT-11 group had a significantly higher CD3+ T cell count versus placebo group. The female subgroup also had a significantly higher CD8+CD28+ T cell count. Although the KT-11 group showed no changes in SARS-CoV-2 immunoglobulin G titer, it had fewer self-reported common cold-like symptoms, particularly fatigue. This pilot study showed that KT-11 affected immune cell profiles, suggesting that the feasibility of a verification study. This trial was registered at UMIN Clinical Trials Registry (UMIN000046991).

Linking warmer nest temperatures to reduced body size in seabird nestlings: Possible mitochondrial bioenergetic and proteomic mechanisms.

Rapid reduction of body size in populations responding to global warming suggests the involvement of temperature-dependent physiological adjustments during growth, such as mitochondrial alterations, in the efficiency of producing metabolic energy, a process that is poorly explored, especially in endotherms. Here, we examined the mitochondrial metabolism and proteomic profile of red blood cells in relation to body size and cellular energetics in nestling shearwaters (Calonectris diomedea) developing at different natural temperatures. We found that nestlings of warmer nests had lighter bodies and smaller beaks at fledging. Despite there was no effect of environmental temperature on cellular metabolic rate, mitochondria had a higher inefficiency in coupling metabolism to allocable energy production, as evidenced by bioenergetic and proteomic analyses. Mitochondrial inefficiency was positively related to cellular stress represented by heat shock proteins, antioxidant enzymes and markers of mitochondrial stress. The observed temperature-related mitochondrial inefficiency was associated with reduced beak size and body mass and was linked to a downregulation of cellular growth factors and growth promoters determining body size. By analyzing the links between environmental temperature, mitochondrial inefficiency and body size we discuss the physiological alterations that free-living birds, and probably other endotherms, need to trigger to cope with a warming world.

Comprehensive Single-Cell Chromatin and Transcriptomic Profiling of Peripheral Immune Cells in Non-Segmental Vitiligo.

Non-segmental vitiligo (NSV) is an autoimmune condition characterized by melanocyte loss. While skin-specific mechanisms are well-studied, systemic immune dysregulation contributing to NSV pathogenesis remains unclear. This study employs a multi-omic single-cell approach to investigate circulating immune cells in NSV, integrating transcriptional and chromatin accessibility data. An integrative scRNA-seq/scATAC-seq analysis was conducted on PBMCs from NSV patients (n=11) and controls (n=5), identifying transcriptional markers, cell-cell interactions, chromatin accessibility, and transcription factor (TF) dynamics. Key findings were validated in an expanded cohort (NSV, n=16; controls, n=9) using spectral flow cytometry, with additional stratification by sex, age, disease activity, severity, and duration. Analysis of 59,192 PBMCs identified 8,204 gene expression markers and 13,925 ATAC peaks across 25 immune cell subtypes. A broadly activated immune response was observed, characterized by cytotoxicity, antigen presentation, cell exhaustion, and stress, predominantly in monocytes, NK cells, CD8+ T cells, and dendritic cells (DCs). Multi-omic integration revealed Th1/Th17 polarization and dysfunctional regulatory T cell (Treg/mTreg) responses. Chromatin accessibility highlighted enriched TF binding sites for FOXO3, SP1, AP1, STAT1/STAT3, IRF1, and IRF4, regulating pathways linked to cytotoxicity, antigen processing, NF-κB, Toll-like receptor, and JAK-STAT signaling.Flow cytometry validated these findings, showing that disease activity and shorter duration were associated with heightened immune dysregulation. Robust TCR activation drove Th1/Th17 polarization and elevated IFN-γ and TNF-α production in CD4+ and CD8+ T cells. CLA+ skin-homing Th1/Th17-polarized CD4+ T cells, CD8+ T cells, and mTregs exhibited persistent activation, marked by basal PD1+ expression. OX40/OX40L-mediated interactions between monocytes and effector T cells amplified inflammation. Regulatory dysfunction, including reduced IL-4 and IL-13 production in mTregs, was prominent in moderate-severe and active disease. This is the first multi-omic single-cell study in PBMCs of NSV patients, revealing systemic immune dysregulation driven by cytotoxicity, antigen presentation, exhaustion, and regulatory failure. Disease severity, activity, and evolution influence these pathways, highlighting the OX40/OX40L axis as a potential therapeutic target to mitigate immune dysregulation and relapse risk.

Comparing the cannabidiol-induced transcriptomic profiles in human and mouse Sertoli cells.

Cannabidiol (CBD), a major cannabinoid found in Cannabis sativa L., has been used in the treatment of seizures associated with Lennox-Gastaut syndrome, Dravet syndrome, and tuberous sclerosis complex. Recently, concerns have been raised regarding the male reproductive toxicity of CBD in animal models, such as monkeys, rats, and mice. In our previous studies, we reported that CBD inhibited cell proliferation in both primary human Sertoli cells and mouse Sertoli TM4 cells. Transcriptomic analysis revealed that in primary human Sertoli cells CBD disrupted DNA replication, cell cycle, and DNA repair, ultimately causing cellular senescence. In this study, we further investigated the molecular changes induced by CBD in mouse Sertoli TM4 cells using RNA-sequencing analyses and compared the transcriptomic profile with that of primary human Sertoli cells. Our findings demonstrated that, unlike in primary human Sertoli cells, CBD did not induce cellular senescence but caused apoptosis in mouse Sertoli TM4 cells. Through transcriptomic data analysis in mouse Sertoli TM4 cells, immune and cellular stress responses were identified. Moreover, transcriptomic comparisons revealed major differences in molecular changes induced by CBD between mouse Sertoli TM4 and primary human Sertoli cells. This suggests that primary human Sertoli cells and mouse Sertoli cells may respond differently to CBD.

Structural Analysis of Cardanol and Its Biological Activities on Human Keratinocyte Cells

Background/Objectives: Cashew nutshell liquid (CNSL) is obtained during the industrial processing of cashew nuts. It contains anacardic acid (2-hydroxy-6-n-pentadecylbenzoic acid) and cardanol (3-n-pentadecylphenol). Therefore, CNSL provides a rich source of phenolic lipids serving as natural antioxidants or precursors for industrial uses. Here, we have analyzed in detail a commercial sample of cardanol by nuclear magnetic resonance (NMR) spectroscopy and its biological activities in the human keratinocyte cell line (HaCaT cells). Methods: The cytotoxic effects, genotoxicity, cell proliferation, and healing properties on HaCaT cells were studied using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) assay, comet assay, proliferation assay, and scratch assay, respectively. Additionally, the modulatory effect of cardanol on the cellular fatty acid profile of HaCaT cells was analyzed by gas chromatography. Results: NMR showed the structure of cardanol as a mixture of the 8′-monoene (42%), the 8′,11′-diene (22%), and the 8′,11′,14′-triene (36%) for the pentadecyl side chain with all double bonds in Z configuration. The cytotoxic effects on HaCaT cells only occurred at high concentrations of cardanol (>10 µg/mL), which caused significant reductions in cell viability. Using the comet assay, a dose-dependent increase in DNA damage was found at concentrations above 10 µg/mL. Scratch assays revealed that cardanol achieved 99% wound closure of HaCaT cells treated with 1 µg/mL cardanol after 48 h. Cardanol at 1 and 0.1 µg/mL significantly enhanced HaCaT cell proliferation and promoted migration, contributing to accelerated wound healing processes. As shown by gas chromatography, 1 µg/mL cardanol increased the total amount of polyunsaturated fatty acids (PUFA), including ω-3, ω-6, and ω-9 fatty acids. Conclusions: Together, these findings suggest that concentrations of <10 µg/mL cardanol are safe and exhibit beneficial biological activities, particularly wound-healing effects on HaCaT cells. Further studies are necessary to explore additional potential applications of cardanol, to refine its formulations for clinical use, and to ensure its safety and action in other target cells and species.