Search Results (5)

Formalin-fixed paraffin-embedded (FFPE) tissues represent an invaluable resource for both basic and clinical research due to their stable preservation of tissue architecture and molecular integrity. Multiplex immunofluorescence (mIF) using tyramide signal amplification (TSA) enables the simultaneous detection of multiple antigens within a single FFPE section. Here, we describe a kit-independent and customizable TSA-based mIF protocol that utilizes commercially available horseradish peroxidase (HRP)-conjugated secondary antibodies and tyramide–fluorophore reagents. The method was applied using FFPE endometriosis tissue, targeting estrogen receptor alpha (ERα), progesterone receptor (PR), α-smooth muscle actin (αSMA), CD20 and CD31. Each staining round was followed by heat-induced epitope removal (HIER) of the bound antibodies while preserving covalently deposited signals. Fluorescence imaging was performed using a multi-channel slide scanner with carefully selected fluorophores to enable optical separation between detection channels. Under the conditions described, the protocol enabled clear visualization of maker-specific staining patterns with preserved tissue morphology. This study provides a practical and flexible TSA-based mIF protocol as a qualitative proof of concept, offering an accessible alternative to commercial kit-based approaches. Further studies will be required to establish quantitative performance and a broader applicability across tissue types. Full article

Background and Objectives: This study explores the immunological landscapes of non-melanoma skin neoplasms (NMSNs), specifically keratoacanthoma (KA), squamous cell carcinoma (SCC), and common warts (VV). Although benign, KA shares histological similarities with low-grade SCC. The tumor microenvironment (TME) plays a key role in tumor progression, affecting angiogenesis, inflammation, and immune evasion. Viral infections, particularly human papillomavirus (HPV), are linked to NMSN development, with various HPV types identified in KA. VV, caused by HPV, serves as a comparative model due to its similar etiopathogenesis. Materials and Methods: This research examines the expression of CTLA4, a critical regulator of T-cell homeostasis, and IFN-γ, a cytokine with immunomodulatory and antiviral effects, in the TME of 41 KA, 37 SCC, and 55 VV samples using multichannel immunofluorescence. Results: The analysis revealed distinct patterns of CTLA4 and IFN-γ expression. SCC exhibited a higher prevalence of CTLA4+IFN-γ+ double-positive lymphocytes, suggesting a more immunosuppressive TME. In contrast, VV showed the highest expression of CTLA4+ cells, while both KA and VV had lower expressions of IFN-γ+ lymphocytes compared to SCC. The increased presence of CTLA4+IFN-γ+ double-positive lymphocytes in SCC suggests that the co-expression of these markers may exert a stronger effect on TME modulation than CTLA4 alone. Conclusions: These findings underscore the potential of immune profiling as a diagnostic tool to differentiate between benign and malignant lesions, such as KA and SCC. Furthermore, the presence of CTLA4+IFN-γ+ lymphocytes, particularly in SCC, may serve as a biomarker for tumor progression and a potential target for future immunotherapy strategies aimed at modulating the immune response in NMSN. Full article

Electromagnetic waves are widely used in both military and civilian fields, which could cause long-term and high-power exposure to certain populations and may pose a health hazard. The aim of this study was to simulate the long-term and high-power working environment of workers using special electromagnetic radiation occupations to clarify the radiation-induced stress response and cardiac damage and thus gain insights into the mechanisms of injuries caused by electromagnetic radiation. In this study, the combination of microwave and stress was an innovative point, aiming to broaden the research direction with regard to the effect and mechanism of cardiac injury caused by radiation. The myocardial structure was observed by optical and transmission electron microscope, mitochondrial function was detected by flow cytometry, oxidative-stress markers were detected by microplate reader, serum stress hormone was detected by radioimmunoassay, and heart rate variability (HRV) was analyzed by multichannel-physiological recorder. The rats were weighed and subjected to an open field experiment. Western blot (WB) and immunofluorescence (IF) were used to detect the expressions and distributions of JNK (c-Jun N-terminal kinase), p-JNK (phosphorylated c-Jun N-terminal kinase), HSF1 (heat shock factor), and NFATc4 (nuclear factor of activated T-cell 4). This study found that radiation could lead to the disorganization, fragmentation, and dissolution of myocardial fibers, severe mitochondrial cavitation, mitochondrial dysfunction, oxidative-stress injury in myocardium, increase to stress hormone in serum, significant changes in HRV, and a slow gain in weight. The open field experiment indicated that the rats experienced anxiety and depression and had decreased exercise capacity after radiation. The expressions of JNK, p-JNK, HSF1, and NFATc4 in myocardial tissue were all increased. The above results suggested that 30 mW/cm² of S-band microwave radiation for 35 min could cause both physiological and psychological stress damage in rats; the damage was related to the activation of the JNK pathway, which provided new ideas for research on protection from radiation. Full article

(1) Background: The immune system has physiological antitumor activity, which is partially mediated by cytotoxic T lymphocytes (CTL). Tumor hypoxia, which is highly prevalent in cancers of the head and neck region, has been hypothesized to inhibit the infiltration of tumors by CTL. In situ data validating this concept have so far been based solely upon the visual assessment of the distribution of CTL. Here, we have established a set of spatial statistical tools to address this problem mathematically and tested their performance. (2) Patients and Methods: We have analyzed regions of interest (ROI) of 22 specimens of cancers of the head and neck region after 4-plex immunofluorescence staining and whole-slide scanning. Single cell-based segmentation was carried out in QuPath. Specimens were analyzed with the endpoints clustering and interactions between CTL, normoxic, and hypoxic tumor areas, both visually and using spatial statistical tools implemented in the R package Spatstat. (3) Results: Visual assessment suggested clustering of CTL in all instances. The visual analysis also suggested an inhibitory effect between hypoxic tumor areas and CTL in a minority of the whole-slide scans (9 of 22, 41%). Conversely, the objective mathematical analysis in Spatstat demonstrated statistically significant inhibitory interactions between hypoxia and CTL accumulation in a substantially higher number of specimens (16 of 22, 73%). It showed a similar trend in all but one of the remaining samples. (4) Conclusion: Our findings provide non-obvious but statistically rigorous evidence of inhibition of CTL infiltration into hypoxic tumor subregions of cancers of the head and neck. Importantly, these shielded sites may be the origin of tumor recurrences. We provide the methodology for the transfer of our statistical approach to similar questions. We discuss why versions of the Kcross and pcf.cross functions may be the methods of choice among the repertoire of statistical tests in Spatstat for this type of analysis. Full article

Background: Epithelial to mesenchymal transition (EMT) promotes therapy resistance in head and neck cancer (HNC) cells. In this study, EMT was quantified in HNC tumor samples by the cellular co-localization of cytokeratin/vimentin, E-cadherin/β-catenin and by Slug expression. Methods: Tissue samples from HNC patients were stained with antibody pairs against cytokeratin/vimentin and E-cadherin/β-catenin. Epithelial–mesenchymal co-localization was quantified using immunofluorescence multichannel image cytometry. Double positivity was confirmed using confocal microscopy. Slug was semi-quantified by 2 specialists and quantified by bright field image cytometry. Results: Tumor samples of 102 patients were investigated. A loss of E-cadherin positive cells (56.9 ± 2.6% vs. 97.9 ± 1.0%; p < 0.0001) and E-cadherin/β-catenin double positive cells (15.4 ± 5.7% vs. 85.4 ± 1.2%; p < 0.0001) was observed in tumor samples. The percentage of Slug positive cells was increased in tumor samples (12.1 ± 3.6% vs. 3.2 ± 2.6%; p = 0.001). Ordinal Slug scores judged by two specialists closely correlated with percentage of Slug-positive cells (Spearman’s rho = 0.81; p < 0.001). Slug score correlated negatively with the percentage of E-cadherin positive cells (r = 0.4; p = 0.006), the percentage of E-cadherin/β-catenin positive cells (r = 0.5; p = 0.001) and positively with cytokeratin/vimentin positive cells (r = 0.4, p = 0.003). Conclusion: EMT can be assessed in HNC tumor probes by cytokeratin/vimentin co-expression and loss of E-cadherin/β-catenin co-expression. Slug score provides a convenient surrogate marker for EMT. Full article