Search Results (8)

Background/Objectives: T-cell lymphomas are often histologically indistinguishable from benign T-cell infiltrates, and diagnosis typically relies on slow, complex, and expensive multiplexed PCR reactions, requiring significant training and experience to interpret them. We aimed to raise highly specific antibodies against the two alternatively used and very similar T-cell receptor beta constant regions, TCRbeta1 and TCRbeta2, encoded by the TRBC1 and TRBC2 gene segments, respectively. We sought to demonstrate the feasibility of detecting TCRbeta1 and TCRbeta2 immunohistochemically in routine clinical (formalin-fixed, paraffin-embedded (FFPE)) tissue sections as a novel diagnostic strategy for T-cell lymphomas. Methods: Recombinant rabbit antibodies were validated using Western blotting and FFPE immunostaining of T-cell leukemia lines. The immunostaining of FFPE tissue containing benign and lymphomatous T-cell populations was undertaken, with corroboration by BaseScope^TM high-sensitivity in situ hybridization and quantitative real-time PCR (Q-PCR). An additional Q-PCR literature review and analysis of publicly available RNAseq data was used to determine the TCRbeta2/TCRbeta1 ratio cut-off to separate benign and malignant T-cell populations. Results: Our TCRbeta1/TCRbeta2 antibody pair gave highly specific FFPE tissue staining. All benign samples analyzed (immunohistochemically, by BaseScope^TM, by Q-PCR, and by RNAseq data analysis) had TCRbeta1/TCRbeta2 or TRBC1/TRBC2 ranges well within the previously published flow cytometric benign range (TCRbeta2/TCRbeta1 = 0.18:1–5.7:1), while samples of T-cell lymphoma did not. One out of thirteen (7.7%) lymphoma samples showed some detectable TCRbeta1/TCRbeta2 protein co-expression, and 4 out of 13 (30.8%) T-cell lymphomas showed a TRBC1/TRBC2 transcript co-expression using BaseScope^TM. Conclusions: Analyzing T-cell monotypia immunohistochemically, analogous to B-cell monotypia (kappa: lambda ratio for B-cell and plasma cell neoplasms), could make the diagnosis of T-cell lymphomas cheaper, quicker, and more accurate. Larger studies are needed to validate our antibodies for clinical use. Full article

Background: Inflammation and immune cell dysfunction are critical facilitators of endometriosis pathophysiology. Macrophages are renowned for stimulating lesion growth, vascularization, innervation, and pain generation. By combining macrophages and endometriotic cells, we determined if resveratrol and its natural analogs can target the immune dysregulation and oxidative imbalance in endometriosis. Methods: After treatment with compounds (5, 10, 25 µM), we evaluated the expression of key inflammatory and oxidative stress markers, cytokines release, and ROS production by applying q-PCR, ELISA, Cytometric Beads Array, and multiplexed fluorogenic staining and flow cytometry analysis with bioimaging. Results: The results showed that endometriosis-related macrophages treated with stilbenes have impaired expression of pro-inflammatory markers (IL6, IL8, IL1B, TNF, CCL2, CXCL10, PTGS2). The effect of resveratrol, pterostilbene, and piceatannol was observed, especially in reducing IL1B, CCL2, and CXCL10 genes up to 3.5-, 5-, and 7.7-fold at 25 µM, respectively. Also, with piceatannol or polydatin exposure, the IL-6 decrease was noticeable. This study reported an antioxidant effect by reducing ROS-positive cells from 96% to 48% by pterostilbene. Results from flow cytometry correlated with the transcript activation of detoxification enzymes (SOD, GPX). Conclusions: Prospects for potential therapy based on regulating the immune microenvironment and reducing the accumulation of free radicals with stilbenes application were described in the article. Full article

Coronary artery disease (CAD) is a long-term inflammatory process, with atherosclerosis as its underlying pathophysiological mechanism. Endothelial dysfunction is the first step towards atherosclerosis, where damaged endothelial cells release large amounts of pro-inflammatory cytokines and mediators, thus promoting vascular inflammation and disease progression. However, the correlation between serum cytokines and CAD severity remains to be defined. Serum samples from patients performing cardiac computed tomography for suspected CAD (n = 75) were analyzed with a multiplex bead-based immunoassay panel for simultaneous assessment of the concentration of 11 cytokines using flow cytometric technology. The analysis showed statistically significant increases in sRAGE, CCL2_MCP1, FLT1, and IL6 levels in CAD patients compared with healthy subjects and a gradual increase trend towards a more severe form of the disease for most cytokines (e.g., sCD40L, FLT1, sRAGE, CCL2-MCP1, TNFα). Lastly, we explored the performance of cytokines in predicting the diagnosis of CAD and found that an increase in IL6 levels will increase the odds of being non-obstructive CAD-positive. In contrast, an increase in CCL2-MCP1 or FLT1 levels will increase the probability of being obstructive CAD-positive. These results suggest that the combination of serum cytokines may contribute to the not-invasive stratification risk for patients with suspected CAD. Full article

Flow cytometry becomes a common method for analysis of spermatozoa quality. Standard sperm characteristics such as viability, acrosome and chromatin integrity, oxidative damage (ROS) etc. can be easily assess in any animal semen samples. Moreover, several fertility-related markers were observed in humans and some other mammals. However, these fertility biomarkers have not been previously studied in ram. The aim of this study was to optimize the flow-cytometric analysis of these standard and novel markers in ram semen. Ram semen samples from Slovak native sheep breeds were analyzed using CASA system for motility and concentration and were subsequently stained with several fluorescent dyes or specific antibodies to evaluate sperm viability (SYBR-14), apoptosis (Annexin V, YO-PRO-1, FLICA, Caspases 3/7), acrosome status (PNA, LCA, GAPDHS), capacitation (merocyanine 540, FLUO-4 AM), mitochondrial activity (MitoTracker Green, rhodamine 123, JC-1), ROS (CM-H₂DCFDA, DHE, MitoSOX Red, BODIPY), chromatin (acridine orange), leukocyte content, ubiquitination and aggresome formation, and overexpression of negative biomarkers (MKRN1, SPTRX-3, PAWP, H3K4me2). Analyzed semen samples were divided into two groups according to viability as indicators of semen quality: Group 1 (viability over 60%) and Group 2 (viability under 60%). Significant (p < 0.05) differences were found between these groups in sperm motility and concentration, apoptosis, acrosome integrity (only PNA), mitochondrial activity, ROS production (except for DHE), leukocyte and aggresome content, and high PAWP expression. In conclusion, several standard and novel fluorescent probes have been confirmed to be suitable for multiplex ram semen analysis by flow cytometry as well as several antibodies have been validated for the specific detection of ubiquitin, PAWP and H3K4me2 in ram spermatozoa. Full article

Galectins are one of the critical players in the tumor microenvironment–tumor crosstalk and the regulation of local immunity. Galectin-9 has been in the limelight in tumor immunology. Galectin-9 possesses its multiplex biological functions both extracellularly and intracellularly, plays a pivotal role in the modulation of adaptive and innate immunity, and induces immune tolerance. NK-92MI cell lines against different malignancies were extensively studied, and recently published trials used genetically chimeric antigen receptor-transfected NK-92MI cells in tumor immunotherapy. Besides the intensive research in tumor immunotherapy, limited information is available on their immune-checkpoint expression and the impact of checkpoint ligands on their effector functions. To uncover the therapeutic potential of modulating Galectin-9-related immunological pathways in NK-cell-based therapy, we investigated the dose-dependent effect of soluble Galectin-9 on the TIM-3 checkpoint receptor and NKG2D, CD69, FasL, and perforin expression of NK-92MI cells. We also examined how their cytotoxicity and cytokine production was altered after Gal-9 treatment and in the presence of different serum supplements using flow cytometric analysis. Our study provides evidence that the Galectin-9/TIM-3 pathway plays an important role in the regulation of NK cell function, and about the modulatory role of Galectin-9 on the cytotoxicity and cytokine production of NK-92MI cells in the presence of different serum supplements. We hope that our results will aid the development of novel NK-cell-based strategies that target Galectin-9/TIM-3 checkpoint in tumors resistant to T-cell-based immunotherapy. Full article

Intraoperative radiotherapy (IORT) displays an increasingly used treatment option for early breast cancer. It exhibits non-inferiority concerning the risk of recurrence compared to conventional external irradiation (EBRT) in suitable patients with early breast cancer. Since most relapses occur in direct proximity of the former tumor site, the reduction of the risk of local recurrence effected by radiotherapy might partially be due to an alteration of the irradiated tumor bed’s micromilieu. Our aim was to investigate if IORT affects the local micromilieu, especially immune cells with concomitant cytokine profile, and if it has an impact on growth conditions for breast cancer cells as well as mammary mesenchymal stromal cells (MSC), the latter considered as a model of the tumor bed stroma.42 breast cancer patients with breast-conserving surgery were included, of whom 21 received IORT (IORT group) and 21 underwent surgery without IORT (control group). Drainage wound fluid (WF) was collected from both groups 24 h after surgery for flow cytometric analysis of immune cell subset counts and potential apoptosis and for multiplex cytokine analyses (cytokine array and ELISA). It served further as a supplement in cultures of MDA-MB 231 breast cancer cells and mammary MSC for functional analyses, including proliferation, wound healing and migration. Furthermore, the cytokine profile within conditioned media from WF-treated MSC cultures was assessed. Flow cytometric analysis showed no group-related changes of cell count, activation state and apoptosis rates of myeloid, lymphoid leucocytes and regulatory T cells in the WF. Multiplex cytokine analysis of the WF revealed group-related differences in the expression levels of several cytokines, e.g., oncostatin-M, leptin and IL-1β. The application of WF in MDA-MB 231 cultures did not show a group-related difference in proliferation, wound healing and chemotactic migration. However, WF from IORT-treated patients significantly inhibited mammary MSC proliferation, wound healing and migration compared to WF from the control group. The conditioned media collected from WF-treated MSC-cultures also exhibited altered concentrations of VEGF, RANTES and GROα. IORT causes significant changes in the cytokine profile and MSC growth behavior. These changes in the tumor bed could potentially contribute to the beneficial oncological outcome entailed by this technique. The consideration whether this alteration also affects MSC interaction with other stroma components presents a promising gateway for future investigations. Full article

The presence and interaction of immune cells in the tumor microenvironment is of significant importance and has a great impact on disease progression and response to therapy. Hence, their identification is of high interest for prognosis and treatment decisions. Besides detailed phenotypic analyses of immune, as well as tumor cells, spatial analyses is an important parameter in the complex interplay of neoplastic and immune cells—especially when moving into focus efforts to develop and validate new therapeutic strategies. Ex vivo analysis of tumor samples by immunohistochemistry staining methods conserves spatial information is restricted to single markers, while flow cytometry (disrupting tissue into single cell suspensions) provides access to markers in larger numbers. Nevertheless, this comes at the cost of scarifying morphological information regarding tissue localization and cell–cell contacts. Further detrimental effects incurred by, for example, tissue digestion include staining artifacts. Consequently, ongoing efforts are directed towards methods that preserve, completely or in part, spatial information, while increasing the number of markers that can potentially be interrogated to the level of conventional flow cytometric methods. Progression in multiplex immunohistochemistry in the last ten years overcame the limitation to 1–2 markers in classical staining methods using DAB with counter stains or even pure chemical staining methods. In this study, we compared the multiplex method Chipcytometry to flow cytometry and classical IHC-P using DAB and hematoxylin. Chipcytometry uses frozen or paraffin-embedded tissue sections stained with readily available commercial fluorophore-labeled antibodies in repetitive cycles of staining and bleaching. The iterative staining approach enables sequential analysis of a virtually unlimited number of markers on the same sample, thereby identifying immune cell subpopulations in the tumor microenvironment in the present study in a humanized mouse melanoma model. Full article

Brain cancer research has been hampered by a paucity of viable clinical tissue of sufficient quality and quantity for experimental research. This has driven researchers to rely heavily on long term cultured cells which no longer represent the cancers from which they were derived. Resection of brain tumors, particularly at the interface between normal and tumorigenic tissue, can be carried out using an ultrasonic surgical aspirator (CUSA) that deposits liquid (blood and irrigation fluid) and resected tissue into a sterile bottle for disposal. To determine the utility of CUSA-derived glioma tissue for experimental research, we collected 48 CUSA specimen bottles from glioma patients and analyzed both the solid tissue fragments and dissociated tumor cells suspended in the liquid waste fraction. We investigated if these fractions would be useful for analyzing tumor heterogeneity, using IHC and multi-parameter flow cytometry; we also assessed culture generation and orthotopic xenograft potential. Both cell sources proved to be an abundant, highly viable source of live tumor cells for cytometric analysis, animal studies and in-vitro studies. Our findings demonstrate that CUSA tissue represents an abundant viable source to conduct experimental research and to carry out diagnostic analyses by flow cytometry or other molecular diagnostic procedures. Full article