Search Results (30)

Intrahepatic cholangiocarcinoma (ICC) is an aggressive liver malignancy with a rising global incidence and limited therapeutic options. Vascular invasion (VI) is a hallmark of advanced disease, correlating with early recurrence and dismal prognosis, yet its tumor microenvironment (TME) drivers remain elusive. We analyzed single-cell RNA sequencing (scRNA-seq) data from 25 ICC samples to systematically characterize the cellular composition and molecular features related to VI. By integrating bulk RNA-seq data, spatial transcriptomics, and multiplex immunofluorescence, we identified a distinct subset of tumor-like cancer-associated fibroblasts (CAFs), termed tCAFs, enriched in VI-positive tumors. Functional enrichment analyses revealed that tCAFs were prominently associated with hypoxia and angiogenesis pathways, findings corroborated by the significant upregulation of tCAF markers (MME and NT5E) in ICC-derived CAFs under hypoxic conditions in vitro. Cell–cell communication analysis and spatial mapping uncovered that tCAFs might promote VI primarily through VEGF signaling interactions with endothelial cells. Integrative bioinformatics and RT-qPCR validation identified three key functional genes in tCAFs: SLC2A1, PTGS2, and PLOD2. In endothelial sprouting assays, pharmacological inhibition of SLC2A1 exerted a pronounced suppressive effect. Consistently, sprouting assays using ICC-derived CAFs with SLC2A1 knockdown confirmed that its downregulation significantly reduced endothelial sprouting capacity. Importantly, administration of the SLC2A1 inhibitor BAY-876 effectively suppressed tumor progression and intrahepatic metastasis in the orthotopic ICC mouse model. Our findings define a VI-associated cellular ecosystem and molecular landscape in ICC, unveiling a novel hypoxia–tCAFs–endothelial cells axis. Furthermore, we identify SLC2A1 as a clinically relevant therapeutic target, offering new insights into tumor VI. Full article

Dermal papilla (DP) cells orchestrate hair follicle growth and cycling by secreting signaling molecules that stimulate follicular epithelial stem cells. The signal peptide CUB-EGF-like domain-containing protein 3 (SCUBE3) was recently identified as a potent anagen stimulator secreted by DP cells. Ageratum houstonianum ethanolic extract (AHE) and its active constituent agerarin exhibit anti-inflammatory properties; however, their effects on hair follicle growth remain unclear. This study aimed to investigate the effects of AHE and agerarin on SCUBE3 expression in primary human DP cells and to elucidate the underlying molecular signaling pathway. Cell viability was assessed by measuring cell confluency. Ex vivo hair growth was analyzed using organ cultures of human hair follicles. Gene and protein expression were determined using reverse transcription-PCR, immunoblot analysis, immunofluorescent staining, tyramide signal amplification-based multiplex immunohistochemistry, and gene promoter-reporter assay in primary human follicle DP cells. In a hair follicle organ culture model, both AHE and agerarin increased the population of the anagen phase and promoted hair shaft elongation. AHE and agerarin significantly upregulated SCUBE3 expression at both the mRNA and protein levels. Mechanistically, AHE and agerarin induced activator protein-1 (AP-1) expression by activating mitogen-activated protein kinase signaling pathways, thereby increasing SCUBE3 gene promoter activity. AHE and agerarin promoted hair follicle growth by upregulating SCUBE3 expression via activation of the MAPK–AP-1 signaling axis. In conclusion, AHE and agerarin may serve as potential therapeutic agents for the prevention and treatment of alopecia (hair loss). Full article

Background: The progression of idiopathic pulmonary fibrosis (IPF) involves distal airway remodeling and bronchiolization; however, the mechanisms driving these changes, particularly the contributions of epithelial stem cells, are not fully understood. K13⁺ hillock cells, normally quiescent in proximal airways, were examined for their potential contribution to IPF pathogenesis. Methods: Spatial immunofluorescence was used to profile K13 expression along the airway axes in IPF and control lungs. Multiplex staining complemented by ex vivo culture assays was used to test expression stability. Single-cell RNA-sequencing (scRNA-seq) data were re-analyzed to identify cell subclusters and pathway enrichments. Meanwhile, cell–cell communication was inferred by using CellChat. Results: K13 was ectopically upregulated in IPF honeycomb cysts, triggering a proximal-like pseudostratified phenotype. This shift was marked by surges in K13⁺ regionally overlapping expression patterns (K5⁺, ~9%; CC10⁺, ~53%; ACE-TUB⁺, ~44%; MUC5AC⁺, ~23%) and a decline in SOX2 expression (~95% to ~64%), with ~70% of residual SOX2^low cells exhibiting elevated K13. Accompanying the expansion of K13⁺ subclusters (basal: 1.8% to 41.5%; club: 10.7% to 31.5%), it was observed that the profibrotic markers (K17, S100A2, LGALS7, IGFBP6) and ontologies related to RNA processing, stress response, and senescence were also enriched. These subclusters also amplified pro-fibrotic signaling (e.g., TGF-β, SEMA3, and GALECTIN-9) associated with epithelial subtypes and HAS1^high fibroblasts. Conclusions: Here, we demonstrate that K13⁺ cell activation is a pivotal event, driving the dysregulated proximalization of distal airways in IPF through fate reprogramming and epithelial-mesenchymal crosstalk. Thus, elucidating these K13-mediated fate dynamics provides a critical framework for understanding IPF pathogenesis. Full article

Background: Scrub typhus is commonly misdiagnosed because of nonspecific clinical features and limited data on the performance of diagnostic tests. This study aimed to evaluate the accuracy of commercially available serological and molecular assays for diagnosing scrub typhus. Methods: Adult patients with suspected scrub typhus who visited a tertiary-care hospital in South Korea from July 2022 to December 2024 were prospectively enrolled. Scrub typhus was confirmed by either a positive real-time polymerase chain reaction (PCR) result for Orientia tsutsugamushi or a ≥ four-fold increase in the O. tsutsugamushi-specific total immunoglobulin (Ig) antibody titer on an immunofluorescence assay (IFA). The diagnostic performances of the serial IFA, an immunochromatography-based rapid diagnostic test (ICT), and multiplex real-time PCR targeting the groEL and 47-kDa genes were compared. Results: Among 159 patients, 81 had scrub typhus and 78 did not. The sensitivity and specificity were 64% and 100% for the serial IFA, 75% and 91% for the ICT, and 95% and 100% for multiplex PCR, respectively. The area under the curve was significantly higher for the ICT (0.819) than for the acute-phase IFA (0.743, p = 0.02). Conclusions: Multiplex real-time PCR provided rapid and highly accurate confirmation of scrub typhus, and an acute-phase ICT may be an alternative to a single acute-phase IFA for early clinical decision-making. Full article

Lung cancer is the leading cause of cancer-related mortality worldwide, with non-small-cell lung cancer (NSCLC) accounting for the majority of cases. Standard tissue biopsies are invasive and unsuitable for repeated monitoring. Liquid biopsy technologies, particularly circulating tumor cell (CTC) analysis, offer a minimally invasive alternative for real-time disease tracking. To address the need for efficient and reproducible CTC isolation, we developed the Cellsway microfluidic CTC enrichment and identification platform, which employs inertial hydrodynamics in a spiral-shaped microfluidic channel comprising hydrofoil-shaped pillars to enable high-throughput, label-free enrichment of CTCs while preserving cell integrity, followed by an optimized CTC identification assay. Analytical performance assessed through spiking experiments using NSCLC cell lines demonstrated recovery rates of 91.9% for H1975 cells and 78.3% for A549 cells. Clinical validation was performed on blood samples from 51 stage IV NSCLC patients. A 7.5 mL volume of peripheral blood was processed with the SwayBox platform, and enriched CTCs were identified through an optimized multiplex immunofluorescence protocol. CTCs were detected in 47% of NSCLC patients, with counts ranging from 0 to 72 cells per 7.5 mL of blood. At a cutoff of 1 CTC per 7.5 mL, the assay achieved a specificity of 95%. Patient-derived CTCs exhibited smaller mean diameters compared to cultured NSCLC cell lines, yet were effectively enriched through hydro-dynamic tuning. These findings demonstrate that the Cellsway platform enables efficient and re-producible CTC isolation with high specificity, supporting its potential utility for clinical monitoring and precision oncology in NSCLC. Full article

Antibody-mediated rejection (ABMR) remains a leading cause of kidney allograft failure, yet the mechanistic roles of innate immune cell subsets such as monocytes and natural killer (NK) cells remain incompletely understood. In this retrospective cohort study, we applied OPAL-based multiplex immunofluorescence (mIF) to human kidney allograft biopsies from 38 recipients with biopsy-proven ABMR (n = 19), T-cell-mediated rejection (TCMR, n = 12), or no rejection (NR, n = 7), enabling spatially resolved quantification of immune subsets in situ. Fluorescence thresholds were pathologist-validated, and co-expression phenotypes were defined using standardized segmentation and spectral unmixing. We observed a significantly higher density of CD1^4+CD11c⁺ monocyte-derived cells in ABMR versus TCMR (p = 0.011), and of cytotoxic CD3⁻PAX8⁻CD16⁺CD57⁺ NK cells in ABMR versus TCMR (p = 0.008), implicating both subsets in ABMR pathogenesis. Spatial clustering of these populations was evident in ABMR biopsies, suggesting organized immune infiltration. A logistic regression model combining both subsets yielded an area under the ROC curve of 0.79 (95% CI: 0.65–0.93), indicating moderate discriminatory power for ABMR. While Cox regression did not reveal statistically significant associations with graft survival, CD3⁻PAX8⁻CD16⁺CD57⁺ cells showed a trend toward increased risk (HR = 2.73, p = 0.09). These findings support a mechanistic role for monocyte and NK cell subsets in ABMR and demonstrate the utility of OPAL mIF for high-resolution immune profiling in human allografts. Our study advances understanding of cellular immune contributors to ABMR and highlights the potential diagnostic value of intragraft mononuclear cell phenotyping. Full article

Ischemic stroke remains a leading cause of death and disability worldwide, with neuroinflammation playing a central role in its pathogenesis. This study aimed to investigate age-related differences in neuroinflammatory responses in the human cerebral cortex following ischemic stroke. Using autopsy-derived brain tissue from 184 patients histological, histochemical, multiplex immunofluorescence, ELISA, and qRT-PCR analyses were conducted to assess neuronal damage, immune cell infiltration and cytokine expression. Morphological examination revealed pannecrosis in infarct cores and moderate inflammatory infiltration in penumbral regions. Multiplex immunofluorescence demonstrated active migration of NK (CD45⁺CD56⁺CD3⁻) and NKT (CD45⁺CD56⁺CD3⁺) cells in young patients, whereas elderly individuals showed a predominance of T lymphocytes (CD45⁺CD56⁻CD3⁺) and a decline in NK/NKT activity. Molecular assays indicated elevated NKG2D receptor expression and higher IFN-γ and proinflammatory cytokine (TNF-α, IL-1β, IL-6) levels in young patients, contrasting with dysregulated cytokine balance and reduced NK cytotoxicity in the elderly. These findings highlight distinct age-dependent immune mechanisms underlying ischemic injury, demonstrating that NK and NKT cells play a crucial role in early neuroinflammation, while aging shifts the balance toward adaptive immune dominance. The study underscores the need to consider patient age when designing neuroprotective and anti-inflammatory therapeutic strategies for ischemic stroke. Full article

Background: The progression of osteosarcoma is closely related to the immune microenvironment. Related studies have found that the RNA-binding motif protein, X-linked (RBMX), plays a regulatory role in modulating the biological characteristics of the tumor microenvironment (TME). However, its regulatory mechanism in osteosarcoma remains unclear. Methods: In this study, the expression of RBMX in osteosarcoma was analyzed using the results of bulk and single-cell transcriptome sequencing of human osteosarcoma. The RBMX knockout cell line was constructed via lentivirus transfection. The mouse subcutaneous implantable tumor model and single-cell transcriptome sequencing analysis revealed the effects of RBMX on the osteosarcoma microenvironment, as verified via multiplex immunofluorescence, flow cytometry, and PCR experiments. Results: Using the TARGET database and multiplex immunofluorescence, we found that RBMX is highly expressed in human osteosarcoma and is associated with poor prognosis. The high expression of RBMX may mediate the immunosuppressive microenvironment of human osteosarcoma. In vitro cell experiments showed that knockout of RBMX significantly inhibited the proliferation of mouse osteosarcoma cells. Through single-cell transcriptome sequencing analysis of subcutaneous implantable tumors in mice, we determined that RBMX deletion substantially elevated the recruitment of cytotoxic CD8⁺T cells within the mouse TME, which was further verified through flow cytometry analysis. Cell coculture assay confirmed that knockout of RBMX significantly enhanced the cytotoxic activity of CD8⁺T cells. Finally, cell communication and in vitro experimental verification revealed that knocking out RBMX might enhance the infiltration of CD8⁺T cells by upregulating histocompatibility 2, K1, and K region (H2-K1) and downregulating thrombospondin 1 (THBS1). Conclusions: This study may provide potential targets for reshaping the immune microenvironment of osteosarcoma and improving its therapeutic efficacy. Full article

Background/Objectives: To compare clinical features of patients with joint hypermobility syndrome/hypermobile Ehlers Danlos Syndrome (JHS/hEDS) who tested positive or negative for anti-nuclear antibodies (ANA), and to determine antibody titers, staining patterns, and reactivity to common nuclear autoantigens. Methods: ANA results were determined by Hep2 immunofluorescence assay. Reactivity to the most common nuclear autoantigens was measured by the Multiplex assay. Clinical manifestations were compared between three subgroups: total ANA+, ANA+ who did not have evidence of systemic autoimmune inflammatory disease (SAID), and ANA−. Results: Of 289 patients, 210 patients had a Beighton score > 5 and were tested for ANA antibodies. One hundred and thirty-one patients had a positive ANA test. Twenty patients in this subgroup were classified as SAID+ while the remaining 111 patients did not meet criteria for any systemic disease. Speckled staining was the most observed pattern in both ANA+SAID+ (75.00%) and ANA+SAID− (72.97%) subgroups. In the latter subgroup, the target of nuclear autoreactivity remained elusive in 80% of patients. The most common clinical manifestations were diffuse arthralgias, myofascial pain, sicca symptoms, Raynaud’s phenomenon, gastrointestinal manifestations, and chronic fatigue. Joint dislocations were observed more commonly in the ANA− subgroup compared to ANA+SAID− patients (30.38% vs. 12.61%, adjusted p < 0.05). Conclusions: Similar clinical characteristics were observed in ANA+ and ANA− subgroups of JHS/hEDS, except for joint dislocations which were more common in the ANA− subgroup. The target of ANA reactivity was unknown in 80% of ANA+JHS/hEDS patients and needs to be determined in future studies. Full article

Triple-negative breast cancer (TNBC) is a clinically and molecularly heterogeneous subtype defined by the absence of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) expression. In this study, tumor specimens from 104 TNBC patients were analyzed to characterize molecular and clinicopathological features and to assess the expression and therapeutic potential of four key surface markers: epidermal growth factor receptor (EGFR), epithelial cell adhesion molecule (EpCAM), tissue factor (TF), and trophoblast cell surface antigen (TROP2). Multiplex immunofluorescence (mIF) demonstrated elevated EGFR and TROP2 expression in the majority of samples. Significant positive correlations were observed between EGFR and TF, as well as between TROP2 and both TF and EpCAM. Expression analyses revealed increased EGFR and TF levels with advancing tumor stage, whereas EpCAM expression declined in advanced-stage tumors. TROP2 and TF expression were significantly elevated in higher-grade tumors. Additionally, EGFR and EpCAM levels were significantly higher in patients with elevated Ki-67 indices. Binding specificity assays using single-chain variable fragment (scFv-SNAP) fusion proteins confirmed robust targeting efficacy, particularly for EGFR and TROP2. These findings underscore the therapeutic relevance of EGFR and TROP2 as potential biomarkers and targets in TNBC. Full article

Background/Objectives: Anti-p200 pemphigoid is a rare and likely underdiagnosed autoimmune blistering disorder. Laminin γ1 and laminin β4 have been implicated as potential target antigens in its pathogenesis. Recently, a novel indirect immunofluorescence assay targeting anti-laminin β4 antibodies has been developed, demonstrating high sensitivity and specificity, and offering a valuable tool for improved diagnosis. Methods: Of the 451 patients, 21 were selected for further laboratory analysis based on medical records. Sera from 10 patients, which showed a positive direct immunofluorescence (DIF) result and negative results in multiplex enzyme-linked immunosorbent assays (ELISAs) and/or mosaic six-parameter indirect immunofluorescence (IIF) for various autoimmune bullous diseases, were tested for the presence of anti-laminin β4 antibodies. Additionally, sera from 11 patients with positive DIF and positive ELISA for antibodies against BP180 and/or BP230 were analyzed. Results: Among the 10 patients with positive DIF and negative ELISA and/or mosaic six-parameter IIF, 6 sera were positive for anti-laminin β4 antibodies. These patients presented with atypical clinical features. In contrast, all 11 sera from patients with both positive DIF and positive ELISA for BP180 and/or BP230 were negative for anti-laminin β4 antibodies. Conclusions: In patients with a positive DIF result but negative ELISA and/or mosaic six-parameter IIF findings, testing for anti-laminin β4 antibodies should be considered. Furthermore, in cases presenting with atypical clinical features—such as acral distribution of lesions, intense pruritus, or erythematous–edematous plaques—the possibility of anti-p200 pemphigoid should be included in the differential diagnosis. Full article

Recent evidence indicates that FAM111B is significantly involved in the progression of various cancers. Nonetheless, the potential pan-cancer implications of FAM111B have not been systematically investigated. In this study, FAM111B’s expression and oncogenic potential were studied using TCGA and GTEx data via GEPIA2, TIMER2.0, and STRING tools. Pathway enrichment analyses with the GO, KEGG, Reactome, and WikiPathways databases were conducted to explore its role in cancer development. The results were validated via multiplex immunofluorescence assays of pancreatic cancer tissues, microarray assays of ovarian cancer tissues, and protein transcriptomics of ovarian cancer cells. The expression levels of FAM111B were elevated in most cancer types and were associated with poor prognostic outcomes. Mechanistically, FAM111B expression was positively correlated with the expression of genes involved in DNA homologous recombination repair and with the infiltration of Th2 CD4+ T cells. These observations were further substantiated in ovarian cancer cell lines and tissue specimens from pancreatic and ovarian cancers. FAM111B functions as a biomarker for the DNA repair pathway and Th2 CD4+ T-cell infiltration in human malignancies. Full article

Background. Currently, it is known that the gut microbiota plays an important role in the functioning of the immune system, and a rebalancing of the bacterial community can arouse complex immune reactions and lead to immune-mediated responses in an organism, in particular, the development of atopic dermatitis (AD). Cytokines and chemokines are regulators of the innate and adaptive immune response and represent the most important biomarkers of the immune system. It is known that changes in cytokine profiles are a hallmark of many diseases, including atopy. However, it remains unclear how the bacterial imbalance disrupts the function of the immune response in AD. Objectives. We attempted to determine the role of gut bacteria in modulating cytokine pathways and their role in atopic inflammation. Methods. We sequenced the 16S rRNA gene from 50 stool samples of children aged 3–12 years who had confirmed atopic dermatitis, and 50 samples from healthy children to serve as a control group. To evaluate the immune status, we conducted a multiplex immunofluorescence assay and measured the levels of 41 cytokines and chemokines in the serum of all participants. Results. To find out whether changes in the composition of the gut microbiota were significantly associated with changes in the level of inflammatory cytokines, a correlation was calculated between each pair of bacterial family and cytokine. In the AD group, 191 correlations were significant (Spearman’s correlation coefficient, p ≤ 0.05), 85 of which were positive and 106 which were negative. Conclusions. It has been demonstrated that intestinal dysbiosis is associated with alterations in cytokine profiles, specifically an increase in proinflammatory cytokine concentrations. This may indicate a systemic impact of these conditions, leading to an imbalance in the immune system’s response to the Th2 type. As a result, atopic conditions may develop. Additionally, a correlation between known AD biomarkers (IL-5, IL-8, IL-13, CCL22, IFN-γ, TNF-α) and alterations in the abundance of bacterial families (Pasteurellaceae, Barnesiellaceae, Eubacteriaceae) was observed. Full article

Previously, NCAPG was identified as a candidate gene associated with sheep growth traits. This study aimed to investigate the direct role of NCAPG in regulating myogenesis in embryonic myoblast cells and to investigate the association between single-nucleotide polymorphisms (SNPs) in its promoter region and sheep growth traits. The function of NCAPG in myoblast proliferation and differentiation was detected after small interfering RNAs (siRNAs) knocked down the expression of NCAPG. Cell proliferation was detected using CCK-8 assay, EdU proliferation assay, and flow cytometry cell cycle analysis. Cell differentiation was detected via cell immunofluorescence and the quantification of myogenic regulatory factors (MRFs). SNPs in the promoter region were detected using Sanger sequencing and genotyped using the improved multiplex ligation detection reaction (iMLDR^®) technique. As a result, a notable decrease (p < 0.01) in the percentage of EdU-positive cells in the siRNA-694-treated group was observed. A significant decrease (p < 0.01) in cell viability after treatment with siRNA-694 for 48 h and 72 h was detected using the CCK-8 method. The quantity of S-phase cells in the siRNA-694 treatment group was significantly decreased (p < 0.01). After interfering with NCAPG in myoblasts during induced differentiation, the relative expression levels of MRFs were markedly (p < 0.05 or p < 0.01) reduced compared with the control group on days 5–7. The myoblast differentiation in the siRNA-694 treatment group was obviously suppressed compared with the control group. SNP1, SNP2, SNP3, and SNP4 were significantly (p < 0.05) associated with all traits except body weight measured at birth and one month of age. SNP5 was significantly (p < 0.05) associated with body weight, body height, and body length in six-month-old sheep. In conclusion, interfering with NCAPG can inhibit the proliferation and differentiation of ovine embryonic myoblasts. SNPs in its promoter region can serve as potential useful markers for selecting sheep growth traits. Full article

Dry eye inflammation is a key step in a vicious circle and needs to be better understood in order to break it. The goals of this work were to, first, characterize alarmins and cytokines released by ocular surface cells in the hyperosmolar context and, second, study the role of NFAT5 in this process. Finally, we studied the potential action of these alarmins in ocular surface epithelial cells and macrophages via RAGE pathways. HCE and WKD cell lines were cultured in a NaCl-hyperosmolar medium and the expression of alarmins (S100A4, S100A8, S100A9, and HMGB1), cytokines (IL6, IL8, TNFα, and MCP1), and NFAT5 were assessed using RT-qPCR, ELISA and multiplex, Western blot, immunofluorescence, and luciferase assays. In selected experiments, an inhibitor of RAGE (RAP) or NFAT5 siRNAs were added before the hyperosmolar stimulations. HCE and WKD cells or macrophages were treated with recombinant proteins of alarmins (with or without RAP) and analyzed for cytokine expression and chemotaxis, respectively. Hyperosmolarity induced epithelial cell inflammation depending on cell type. NFAT5, but not RAGE or alarmins, participated in triggering epithelial inflammation. Furthermore, the release of alarmins induced macrophage migration through RAGE. These in vitro results suggest that NFAT5 and RAGE have a role in dry eye inflammation. Full article