Search

For precision cancer radiotherapy, high linear energy transfer (LET) particle irradiation offers a substantial advantage over photon-based irradiation. In contrast to the sparse deposition of low-density energy by χ- or γ-rays, particle irradiation causes focal DNA damage through high-density energy deposition along the particle tracks. This is characterized by the formation of multiple damage sites, comprising localized clustered patterns of DNA single- and double-strand breaks as well as base damage. These clustered DNA lesions are key determinants of the enhanced relative biological effectiveness (RBE) of energetic nuclei. However, the search for a fingerprint of particle exposure remains open, while the mechanisms underlying the induction of chromothripsis-like chromosomal rearrangements by high-LET radiation (resembling chromothripsis in tumors) await to be elucidated. In this work, we investigate the transformation of clustered DNA lesions into chromosome fragmentation, as indicated by the induction and post-irradiation repair of chromosomal damage under the dynamics of premature chromosome condensation in G0 human lymphocytes. Specifically, this study provides, for the first time, experimental evidence that particle irradiation induces localized shattering of targeted chromosome domains. Yields of chromosome fragments and shattered domains are compared with those generated by γ-rays; and the RBE values obtained are up to 28.6 for α-particles (92 keV/μm), 10.5 for C-ions (295 keV/μm), and 4.9 for protons (28.5 keV/μm). Furthermore, we test the hypothesis that particle radiation-induced persistent clustered DNA lesions and chromatin decompaction at damage sites evolve into localized chromosome shattering by subsequent chromatin condensation in a single catastrophic event—posing a critical risk for random rejoining, chromothripsis, and carcinogenesis. Consistent with this hypothesis, our results highlight the potential use of shattered chromosome domains as a fingerprint of high-LET exposure, while conforming to the new model we propose for the mechanistic origin of chromothripsis-like rearrangements. [ABSTRACT FROM AUTHOR]

Simple Summary: The extracellular matrix surrounding or infiltrating tumor tissues, tumor cells, endothelial cells, immune cells, fibroblasts, macrophages, as well as soluble molecules like cytokines and growth factors secreted by these cells, constitute the complex biological structure known as the tumor microenvironment (TME). The cellular and non-cellular components of TME have a role in the development of tumors and the immune response, which offers novel insights for targeted therapies. Depleting existing cells, stopping them from being attracted to tumor locations, and reprogramming them into antitumor subtypes are the three primary categories of therapeutic approaches. TME exhibits complicated connections between Hodgkin/Reed–Sternberg cells and microenvironment and plays a crucial role in classical Hodgkin lymphoma (CHL) as well. Numerous studies have demonstrated that an extensive understanding of the CHL microenvironment, including the identification of all cellular components and variables implicated in the pathogenesis, is essential for improving prognostic stratification and developing innovative targeted treatments. Although classical Hodgkin lymphoma (CHL) is typically curable, 15–25% of individuals eventually experience a relapse and pass away from their disease. In CHL, the cellular microenvironment is constituted by few percent of H/RS (Hodgkin/Reed–Sternberg) tumor cells surrounded from a heterogeneous infiltration of inflammatory cells. The interplay of H/RS cells with other immune cells in the microenvironment may provide novel strategies for targeted immunotherapies. In this paper we analyzed the microenvironment content in CHL patients with responsive disease (RESP) and patients with relapsed/refractory disease to treatment (REL). Our results indicate the increase of CD68+ and CD163+ macrophages, the increase of PDL-1+ cells and of CD34+ microvessels in REL patients respective to RESP patients. In contrast we also found the decrease of CD3+ and of CD8+ lymphocytes in REL patients respective to RESP patients. Finally, in REL patients our results show the positive correlation between CD68+ macrophages and PDL-1+ cells as well as a negative correlation between CD163+ and CD3+. [ABSTRACT FROM AUTHOR]

Simple Summary: Systemic inflammation and changes in the inflammatory status are frequent features of lung cancer. There is a close interconnection between cancer development and the clinical, general, and inflammatory status of patients. In this paper, we evaluate a large panel of inflammatory indexes in patients who underwent lung resection for NSCLC lung cancer; we show that pre-operative inflammatory status strongly influences long-term prognosis in patients affected by NSCLC and undergoing surgery. Background (1): Our goal was to investigate if and how pre-operative inflammatory status can influence the long-term prognosis of patients undergoing lung surgery for cancer. Materials and Methods (2): This prospective observational study includes the agreement of all operable patients to the study, who were referred to our department between 1 January 2017 and 30 December 2018. The inflammatory pre-operative status of the patients was investigated by calculating albumin, CPR (c-protein reactive), complete blood count (neutrophils, lymphocytes, platelets, hemoglobin), and some other indexes referring to inflammatory status, namely the HALP amalgamated index, platelet-to-lymphocyte ratio (PLR), neutrophil-to-lymphocytes ratio (NLR), systemic immune-inflammation index (SII), and advanced lung cancer inflammation Index (ALI). The follow-up ended in November 2021. Patient overall survival was assessed using the Kaplan–Meier method. The log-rank test was used to compare survival rates. Variables significantly associated with survival at univariate analysis were entered int Cox multivariate analysis (stepwise mode) to assess their independent character. Hazard ratios and their 95% confidence intervals were calculated. Variables associated with p < 0.05 were considered significative. Results (3): We enrolled 257 patients in our study. The overall survival of the cohort was as follows: 1 year, 96.1%; 3 year, 81.3%; and 4 year, 74.2%. Univariate analysis showed risk factors for overall survival as follows: Thoracoscore ≥ 2 (p = 0.002); histology (p = 0.002); HALP < 32.2 (p = 0.0002); SII ≥ 808.9 (p = 0.0004); ALI < 34.86 (p = 0.0005); NLr ≥ 2.29 (p = 0.01); hemoglobin < 13 g/dl (p = 0.01); PLR ≥ 196.1 (p = 0.005); pN+ (p < 0.0001); pleural invasion (p = 0.0002); and presence of vascular or lymphatic tumor emboli (p = 0.0002). Multivariate Cox analysis (stepwise model) identified Thoracoscore ≥ 2 (p = 0.02); histology, HALP < 32.2 (p = 0.004), and pN (p < 0.0001) as independent predictors of death. Conclusion (4): Pre-operative inflammatory status strongly influences long-term prognosis in patients affected by NSCLC and undergoing surgery. [ABSTRACT FROM AUTHOR]

Dengue fever, also known as break-bone fever, can be life-threatening. Caused by DENV, an RNA virus from the Flaviviridae family, dengue is currently a globally important public health problem. The clinical methods available for dengue diagnosis require skilled supervision. They are manual, time-consuming, labor-intensive, and not affordable to common people. This paper describes a method that can support clinicians during dengue diagnosis. It is proposed to automate the peripheral blood smear (PBS) examination using Artificial Intelligence (AI) to aid dengue diagnosis. Nowadays, AI, especially Machine Learning (ML), is increasingly being explored for successful analyses in the biomedical field. Digital pathology coupled with AI holds great potential in developing healthcare services. The automation system developed incorporates a blob detection method to detect platelets and thrombocytopenia from the PBS images. The results achieved are clinically acceptable. Moreover, an ML-based technique is proposed to detect dengue from the images of PBS based on the lymphocyte nucleus. Ten features are extracted, including six morphological and four Gray Level Spatial Dependance Matrix (GLSDM) features, out of the lymphocyte nucleus of normal and dengue cases. Features are then subjected to various popular supervised classifiers built using a ten-fold cross-validation policy for automated dengue detection. Among all the classifiers, the best performance was achieved by Support Vector Machine (SVM) and Decision Tree (DT), each with an accuracy of 93.62%. Furthermore, 1000 deep features extracted using pre-trained MobileNetV2 and 177 textural features extracted using Local binary pattern (LBP) from the lymphocyte nucleus are subjected to feature selection. The ReliefF selected 100 most significant features are then fed to the classifiers. The best performance was attained using an SVM classifier with 95.74% accuracy. With the obtained results, it is evident that this proposed approach can efficiently contribute as an adjuvant tool for diagnosing dengue from the digital microscopic images of PBS. [ABSTRACT FROM AUTHOR]

Simple Summary: The High Throughput Truthing project aims to develop a dataset of stromal tumor-infiltrating lymphocytes (sTILs) density evaluations in hematoxylin and eosin-stained invasive breast cancer specimens fit for a regulatory purpose. After completion of the pilot study, the analysis demonstrated inconsistencies and gaps in the provided training to pathologists. Select regions of interest (ROIs) were reviewed by an expert panel, who provided annotations and commentary on the challenges of the sTILs assessment. We used these annotations to develop a training document and reference standard for new training materials. These materials will train crowd-sourced pathologists to help create an algorithm validation dataset and contribute to sTILs evaluations in clinical practice. The High Throughput Truthing project aims to develop a dataset for validating artificial intelligence and machine learning models (AI/ML) fit for regulatory purposes. The context of this AI/ML validation dataset is the reporting of stromal tumor-infiltrating lymphocytes (sTILs) density evaluations in hematoxylin and eosin-stained invasive breast cancer biopsy specimens. After completing the pilot study, we found notable variability in the sTILs estimates as well as inconsistencies and gaps in the provided training to pathologists. Using the pilot study data and an expert panel, we created custom training materials to improve pathologist annotation quality for the pivotal study. We categorized regions of interest (ROIs) based on their mean sTILs density and selected ROIs with the highest and lowest sTILs variability. In a series of eight one-hour sessions, the expert panel reviewed each ROI and provided verbal density estimates and comments on features that confounded the sTILs evaluation. We aggregated and shaped the comments to identify pitfalls and instructions to improve our training materials. From these selected ROIs, we created a training set and proficiency test set to improve pathologist training with the goal to improve data collection for the pivotal study. We are not exploring AI/ML performance in this paper. Instead, we are creating materials that will train crowd-sourced pathologists to be the reference standard in a pivotal study to create an AI/ML model validation dataset. The issues discussed here are also important for clinicians to understand about the evaluation of sTILs in clinical practice and can provide insight to developers of AI/ML models. [ABSTRACT FROM AUTHOR]

Simple Summary: In patients with relapsed or metastasized squamous cell cancer of the head and neck (R/M SCCHN), the PD-L1 Combined Positive Score (CPS) is currently the only predictive biomarker for treatment with anti-PD-1 agents. However, ambiguous results have been determined regarding the overall response rates of immunotherapeutic agents based on PD-L1 status, which may be partially attributed to spatiotemporal heterogeneity. Furthermore, tumor-infiltrating lymphocytes (TILs) have proven to be of significant prognostic value, yet lack of a standardized method for quantification impedes their integration into the current armamentarium of biomarkers in SCCHN. In this paper, concordance of PD-L1 CPS and stromal TILs was investigated in different paired samples of SCCHN subtypes. The results were then linked to well-known clinicopathological variables and prognosis. Background: The clinical significance of tumor-infiltrating lymphocytes (TILs) and programmed cell death-ligand 1 (PD-L1) expression has been thoroughly researched in squamous cell carcinoma of the head and neck (SCCHN). To address the impact of intra- and intertumoral heterogeneity in these biomarkers, we explored the concordance of PD-L1 combined positive score (CPS) and stromal TILs in different paired tissue sample types, while evaluating their internal relationship and prognostic impact. Methods: A total of 165 tissue blocks from 80 SCCHN patients were reviewed for TILs and PD-L1 CPS. Concordance between paired tissue samples was evaluated, and their association with several clinicopathological variables, overall survival (OS), and disease-free survival (DFS) was determined. Results: Biopsies and paired resection material were severely discordant in 39% and 34% of samples for CPS and TIL count, respectively, of which CPS was underscored in 27% of biopsies. In paired primary tumor–metastatic lesions, the disagreement was lower for CPS (19%) but not for TIL count (44%). PD-L1 CPS was correlated with prolonged OS when calculated from tissue acquirement, while extended OS and DFS were observed for high TIL density. Conclusion: Intertumoral and, especially, intratumoral heterogeneity were confounding factors when determining PD-L1 CPS and TIL count on paired tissue samples, indicating the increasing necessity of assessing both biomarkers on representative tissue material. Although TILs hold valuable prognostic information in SCCHN, the robustness of PD-L1 as a biomarker in SCCHN remains ambiguous. [ABSTRACT FROM AUTHOR]

In this paper, we examined the effects of melittin, a bee venom membrane-active peptide, on mitochondrial respiration and cell viability of healthy human lymphocytes (HHL) and Jurkat cells, as well as on lymphoblasts from acute human T cell leukemia. The viability of melittin-treated cells was related to changes in O 2 consumption and in the respiratory control index (RCI) of mitochondria isolated from melittin-pretreated cells as well as of mitochondria first isolated from cells and then directly treated with melittin. It was shown that melittin is three times more cytotoxic to Jurkat cells than to HHL, but O 2 consumption and RCI values of mitochondria from both cell types were equally affected by melittin when melittin was directly added to mitochondria. To elucidate the molecular mechanism of melittin's cytotoxicity to healthy and cancer cells, the effects of melittin on lipid-packing and on the dynamics in model plasma membranes of healthy and cancer cells, as well as of the inner mitochondrial membrane, were studied by EPR spin probes. The affinity of melittin binding to phosphatidylcholine, phosphatidylserine, phosphatidic acid and cardiolipin, and binding sites of phospholipids on the surface of melittin were studied by 31 P-NMR, native PAGE and AutoDock modeling. It is suggested that the melittin-induced decline of mitochondrial bioenergetics contributes primarily to cell death; the higher cytotoxicity of melittin to cancer cells is attributed to its increased permeability through the plasma membrane.

Nasu-Hakola Disease (NHD) is a recessively inherited systemic leukodystrophy disorder characterized by a combination of frontotemporal presenile dementia and lytic bone lesions. NHD is known to be genetically related to a structural defect of TREM2 and DAP12, two genes that encode for different subunits of the membrane receptor signaling complex expressed by microglia and osteoclast cells. Because of its rarity, molecular or proteomic studies on this disorder are absent or scarce, only case reports based on neuropsychological and genetic tests being reported. In light of this, the aim of this paper is to provide evidence on the potential of a label-free proteomic platform based on the Multidimensional Protein Identification Technology (MudPIT), combined with in-house software and on-line bioinformatics tools, to characterize the protein expression trends and the most involved pathways in NHD. The application of this approach on the Lymphoblastoid cells from a family composed of individuals affected by NHD, healthy carriers and control subjects allowed for the identification of about 3000 distinct proteins within the three analyzed groups, among which proteins anomalous to each category were identified. Of note, several differentially expressed proteins were associated with neurodegenerative processes. Moreover, the protein networks highlighted some molecular pathways that may be involved in the onset or progression of this rare frontotemporal disorder. Therefore, this fully automated MudPIT platform which allowed, for the first time, the generation of the whole protein profile of Lymphoblastoid cells from Nasu-Hakola subjects, could be a valid approach for the investigation of similar neurodegenerative diseases.

Outdoor air pollution is associated with respiratory infections and allergies, yet the role of innate lymphoid cells (ILCs) in pathogen containment and airway hyperresponsiveness relevant to effects of air pollutants on ILCs is poorly understood. We conducted a systematic review to evaluate the available evidence on the effect of outdoor air pollutants on the lung type 1 (ILC1) and type 2 ILCs (ILC2) subsets. We searched five electronic databases (up to Dec 2018) for studies on the effect of carbon monoxide (CO), sulfur dioxide (SO 2 ), nitrogen dioxide (NO 2 ), diesel exhaust particles (DEP), ozone (O 3 ), and particulate matter (PM) on respiratory ILCs. Of 2209 identified citations, 22 full-text papers were assessed for eligibility, and 12 articles describing experimental studies performed in murine strains (9) and on human blood cells (3) were finally selected. Overall, these studies showed that exposure to PM, DEP, and high doses of O 3 resulted in a reduction of interferon gamma (IFN-γ) production and cytotoxicity of ILC1. These pollutants and carbon nanotubes stimulate lung ILC2s, produce high levels of interleukin (IL)-5 and IL-13, and induce airway hyperresponsiveness. These findings highlight potential mechanisms by which human ILCs react to air pollution that increase the susceptibility to infections and allergies.

IL-15 is a homeostatic cytokine for human T and NK cells. However, whether other cytokines influence the effect of IL-15 is not known. We studied the impact that IL-10, TGF-β, IL-17A, and IFN-γ have on the IL-15-induced proliferation of human T cells and the expression of HLA class I (HLA-I) molecules. Peripheral blood lymphocytes (PBLs) were labeled with CFSE and stimulated for 12 days with IL-15 in the absence or presence of the other cytokines. The proportion of proliferating T cells and the expression of cell surface HLA-I molecules were analyzed using flow cytometry. The IL-15-induced proliferation of T cells was paralleled by an increase in the expression of HC-10-reactive HLA-I molecules, namely on T cells that underwent ≥5–6 cycles of cell division. It is noteworthy that the IL-15-induced proliferation of T cells was potentiated by IL-10 and TGF-β but not by IL-17 or IFN-γ and was associated with a decrease in the expression of HC-10-reactive molecules. The cytokines IL-10 and TGF-β potentiate the proliferative capacity that IL-15 has on human T cells in vitro, an effect that is associated with a reduction in the amount of HC-10 reactive HLA class I molecules induced by IL-15. [ABSTRACT FROM AUTHOR]

The hypothalamic–pituitary–adrenal axis is known to be involved in the pathogenesis of epilepsy and psychiatric disorders. Epileptic seizures (ESs) and psychogenic non-epileptic seizures (PNESs) are frequently differentially misdiagnosed. This study aimed to evaluate changes in serum cortisol and prolactin levels after ESs and PNESs as possible differential diagnostic biomarkers. Patients over 18 years with ESs (n = 29) and PNESs with motor manifestations (n = 45), captured on video-EEG monitoring, were included. Serum cortisol and prolactin levels as well as hemograms were assessed in blood samples taken at admission, during the first hour after the seizure, and after 6, 12, and 24 h. Cortisol and prolactine response were evident in the ES group (but not the PNES group) as an acute significant increase within the first hour after seizure. The occurrence of seizures in patients with ESs and PNESs demonstrated different circadian patterns. ROC analysis confirmed the accuracy of discrimination between paroxysmal events based on cortisol response: the AUC equals 0.865, with a prediction accuracy at the cutoff point of 376.5 nmol/L 0.811 (sensitivity 86.7%, specificity 72.4%). Thus, assessments of acute serum cortisol response to a paroxysmal event may be regarded as a simple, fast, and minimally invasive laboratory test contributing to differential diagnosis of ESs and PNESs. [ABSTRACT FROM AUTHOR]

Simple Summary: Recessive dystrophic epidermolysis bullosa (RDEB) is a rare and severe hereditary skin disease characterized by skin and mucosa fragility. RDEB patients are predisposed to the development of life-threatening cutaneous squamous cell carcinoma (SCC). In this study, we investigated the immune profiles of SCCs occurring in a cohort of RDEB patients and compared them with clinical, histopathological and prognostic features. We describe two distinct clinical outcomes in RDEB patients with cutaneous SCCs. A majority of RDEB patients had local and not rapidly life-threatening SCC, while others developed early aggressive and metastatic SCCs. The high-risk primary RDEB-SCC was associated with a tumor microenvironment displaying a higher neutrophil-to-lymphocyte infiltration ratio. Increased levels of citrullinated histone H3, a marker of neutrophil extracellular traps (NET), were detected in tumoral skin and in the serum of RDEB patients with high-risk primary SCC. Recessive dystrophic epidermolysis bullosa (RDEB) is a rare severe hereditary skin disease characterized by skin and mucosa fragility, resulting in blister formation. The most severe complication in RDEB patients is the development of cutaneous squamous cell carcinoma (SCC), leading to premature death. There is a great deal of evidence suggesting a permissive tumor microenvironment (TME) as a driver of SCC development in RDEB patients. In a cohort of RDEB patients, we characterized the immune profiles of RDEB-SCCs and compared them with clinical, histopathological, and prognostic features. RDEB-SCCs were subdivided into four groups based on their occurrence (first onset or recurrences) and grading according to clinical, histopathological parameters of aggressiveness. Thirty-eight SCCs from 20 RDEB patients were analyzed. Five RDEB patients experienced an unfavorable course after the diagnosis of the first SCC, with early recurrence or metastasis, whereas 15 patients developed multiple SCCs without metastasis. High-risk primary RDEB-SCCs showed a higher neutrophil-to-lymphocyte ratio in the tumor microenvironment and an increased proportion of neutrophil extracellular traps (NETs). Additionally, citrullinated histone H3, a marker of NETs, was increased in the serum of RDEB patients with high-risk primary SCC, suggesting that this modified form of histone H3 may serve as a potential blood marker of unfavorable prognosis in RDEB-SCCs. [ABSTRACT FROM AUTHOR]

Background and Objectives: Sepsis involves a dysregulated host response, characterized by simultaneous immunosuppression and hyperinflammation. Initially, there is the release of pro-inflammatory factors and immune system dysfunction, followed by persistent immune paralysis leading to apoptosis. This study investigates sepsis-induced apoptosis and its pathways, by assessing changes in PD-1 and PD-L1 serum levels, CD4+ and CD8+ T cells, and Sequential Organ Failure Assessment (SOFA) and Acute Physiology and Chronic Health Evaluation (APACHE II) severity scores. Materials and Methods: This prospective, observational, single-centre study enrolled 87 sepsis patients admitted to the intensive care unit at the County Emergency Clinical Hospital in Târgu Mureș, Romania. We monitored the parameters on day 1 (the day sepsis or septic shock was diagnosed as per the Sepsis-3 Consensus) and day 5. Results: Our study found a statistically significant variation in the SOFA score for the entirety of the patients between the studied days (p = 0.001), as well as for the studied patient groups: sepsis, septic shock, survivors, and non-survivors (p = 0.001, p = 0.003, p = 0.01, p = 0.03). On day 1, we found statistically significant correlations between CD8+ cells and PD-1 (p = 0.02) and PD-L1 (p = 0.04), CD4+ and CD8+ cells (p < 0.0001), SOFA and APACHE II scores (p < 0.0001), and SOFA and APACHE II scores and PD-L1 (p = 0.001 and p = 0.01). On day 5, we found statistically significant correlations between CD4+ and CD8+ cells and PD-L1 (p = 0.03 and p = 0.0099), CD4+ and CD8+ cells (p < 0.0001), and SOFA and APACHE II scores (p < 0.0001). Conclusions: The reduction in Th CD4+ and Tc CD8+ lymphocyte subpopulations were evident from day 1, indicating that apoptosis is a crucial factor in the progression of sepsis and septic shock. The increased expression of the PD-1/PD-L1 axis impairs costimulatory signalling, leading to diminished T cell responses and lymphopenia, thereby increasing the susceptibility to nosocomial infections. [ABSTRACT FROM AUTHOR]

To better understand the mechanism of action of the compounds in the ethanolic extracts of J. nigra leaves and green husks, their binding to CT-DNA was investigated. This study was conducted to elucidate the in vitro protective effect of extracts against chromosomal damage in mitogen-induced human lymphocytes and investigate the possible application of selec+ted extracts as a natural source of polyphenolic compounds. Using HPLC-MS analysis, 103 different compounds were identified as having a higher number of active species, which is consistent with their activity. The frequency of micronuclei (MN) was scored in binucleated cells, and the nuclear proliferation index was calculated. Cyclic voltammetry experiments demonstrate that the nature of the interaction between extracts and CT-DNA is a synergy of electrostatic and intercalative modes, where leaves extracts showed a higher ability to bind to DNA. Extracts showed excellent antioxidant activity. At a concentration of only 4 µg/mL, extract of J. nigra leaves and the green husks reduced the incidence of MN by 58.2% and 64.5%, respectively, compared to control cell cultures. [ABSTRACT FROM AUTHOR]

Glufosinate-ammonium (GLA), an organophosphate herbicide, is released at high concentrations in the environment, leading to concerns over its potential genotoxic effects. However, few articles are available in the literature reporting the possible cellular and nuclear effects of this compound. We assessed, by in vitro and in vivo micronucleus assays, the genotoxicity of GLA on cultured human lymphocytes and Lymnaea stagnalis hemocytes at six concentrations: 0.010 (the established acceptable daily intake value), 0.020, 0.050, 0.100, 0.200, and 0.500 µg/mL. In human lymphocytes, our results reveal a significant and concentration-dependent increase in micronuclei frequency at concentrations from 0.100 to 0.500 μg/mL, while in L. stagnalis hemocytes, significant differences were found at 0.200 and 0.500 μg/mL. A significant reduction in the proliferation index was observed at all tested concentrations, with the only exception of 0.010 μg/mL, indicating that the exposure to GLA could lead to increased cytotoxic effects. In L. stagnalis, a significant reduction in laid eggs and body growth was also observed at all concentrations. In conclusion, we provided evidence of the genomic and cellular damage induced by GLA on both cultured human lymphocytes and a model organism's hemocytes; in addition, we also demonstrated its effects on cell proliferation and reproductive health in L. stagnalis. [ABSTRACT FROM AUTHOR]

In patients with COVID-19, broad panels of immune checkpoint molecules (ICPMs) and the purinergic signaling have not been studied in parallel. We aimed to perform in-depth immunophenotyping of major cell subsets present in human peripheral blood of COVID-19 patients and controls using PD1, TIM3, LAG3, TIGIT, and CD200R, as well as CD39, as markers for the purinergic signaling pathway. We studied 76 COVID-19 patients and 12 healthy controls using peripheral blood mononuclear cells on flow cytometry. Univariable and multivariable statistics were performed. All ICPMs studied were significantly overexpressed on different cell subsets of COVID-19 patients when compared with healthy controls. Elevated lactate dehydrogenase; C-reactive protein; age; and high expression of CD45+, CD39+CD45+, TIM3+CD39+CD4+CD45+, and TIM3+CD39+CD8+CD3+CD4+ cells were significantly associated with severe COVID-19. On multivariable analysis, however, only high expression of CD39+CD45+ (OR 51.4, 95% CI 1.5 to 1763) and TIM3+CD39+CD4+CD3+CD45+ (OR 22.6, 95% CI 1.8 to 277) cells was an independent predictor for severe COVID-19. In conclusion, numerous ICPMs are overexpressed in COVID-19 patients when compared with healthy controls, suggesting a pathophysiological role of these molecules in SARS-CoV-2 infection. However, only TIM3 in co-expression with CD39 remained as a significant independent prognostic ICPM on multivariable analysis. The flow cytometric evaluation of TIM3+CD39+CD4+CD3+CD45+, as well as CD39+CD45+, is a powerful tool for the prognostication of COVID-19 patients on hospital admission. [ABSTRACT FROM AUTHOR]

Simple Summary: Glioblastoma is a lethal primary brain tumor, and so far, immunotherapeutic strategies have not significantly improved patients' prognosis, both in newly diagnosed and recurrent settings. Understanding the features of the immune environment of the central nervous system is crucial to designing new treatment strategies able to counteract site-specific immunosuppressive and pro-tumorigenic factors. Here, we review next-generation immunomodulating therapeutic strategies such as immune check-point blockade, vaccinations, and adoptive cell therapies, aiming to re-shape the tumor microenvironment and restore active anti-tumor immunity. The door for immunotherapeutic strategies in glioblastoma treatment is not completely closed; researchers should test combinatorial treatments and design trials with solid translational analyses to gain more close and deep insight into on-treatment modifications of the tumor microenvironment. Glioblastoma (GBM) is the most aggressive and lethal primary brain tumor, bearing a survival estimate below 10% at five years, despite standard chemoradiation treatment. At recurrence, systemic treatment options are limited and the standard of care is not well defined, with inclusion in clinical trials being highly encouraged. So far, the use of immunotherapeutic strategies in GBM has not proved to significantly improve patients' prognosis in the treatment of newly diagnosed GBM, nor in the recurrent setting. Probably this has to do with the unique immune environment of the central nervous system, which harbors several immunosuppressive/pro-tumorigenic factors, both soluble (e.g., TGF-β, IL-10, STAT3, prostaglandin E2, and VEGF) and cellular (e.g., Tregs, M2 phenotype TAMs, and MDSC). Here we review the immune composition of the GBMs microenvironment, specifically focusing on the phenotype and function of the T cell compartment. Moreover, we give hints on the therapeutic strategies, such as immune checkpoint blockade, vaccinations, and adoptive cell therapy, that, interacting with tumor-infiltrating lymphocytes, might both target in different ways the tumor microenvironment and potentiate the activity of standard therapies. The path to be followed in advancing clinical research on immunotherapy for GBM treatment relies on a twofold strategy: testing combinatorial treatments, aiming to restore active immune anti-tumor responses, tackling immunosuppression, and additionally, designing more phase 0 and window opportunity trials with solid translational analyses to gain deeper insight into the on-treatment shaping of the GBM microenvironment. [ABSTRACT FROM AUTHOR]

The objective of this study was to investigate regulatory T cells (Tregs) and monocytes; specifically, the expression of CTLA-4 (CD152) and FOXP3+ in CD4+CD25+ Tregs and the expression of CD40+ and CD192+ monocyte subpopulations in subjects with primary progressive multiple sclerosis (PPMS). Immunological analysis was conducted on peripheral blood samples collected from the 28 PPMS subjects (15 treated with ocrelizumab and 13 untreated PPMS subjects) and 10 healthy control subjects (HCs). The blood samples were incubated with antihuman CD14, CD16, CD40, and CD192 antibodies for monocytes and antihuman CD4, CD25, FOXP3, and CTLA-4 antibodies for lymphocytes. The study results showed that in comparison to HCs both ocrelizumab treated (N = 15) and untreated (N = 13) PPMS subjects had significantly increased percentages of CTLA-4+ and FOXP3+ in CD4+CD25+ Tregs. Further, ocrelizumab treated PPMS subjects, compared to the untreated ones, had significantly decreased percentages of CD192+ and CD40+ nonclassical monocytes. Increased percentages of CTLA-4+ and FOXP3+ in CD4+CD25+ Tregs in both ocrelizumab treated and untreated PPMS subjects indicates the suppressive (inhibitory) role of Tregs in abnormal immune responses in PPMS subjects. Decreased percentages of CD40+, CD192+, and CD14+CD16++ monocytes for treated compared to untreated PPMS subjects suggests a possible role for ocrelizumab in dampening CNS inflammation. [ABSTRACT FROM AUTHOR]

A correction to the article "Characterization of the Microenvironment of Nodular Lymphocyte Predominant Hodgkin Lymphoma," published in the "International Journal of Molecular Sciences," is presented.

This meta-analysis aimed to evaluate the prognostic value of tumor-infiltrating lymphocytes (TILs) and programmed death-ligand 1 (PD-L1), their associations with the clinicopathological characteristics, and the association between their levels in patients with triple-negative breast cancer (TNBC). PubMed, EMBASE, Scopus, ProQuest, Web of Science, and Cochrane Library databases were searched to obtain the relevant papers. Seven studies with 1152 patients were included in this study. Like the level of TILs, there were no significant associations between PD-L1 expression and tumor size, tumor stage, lymph node metastasis, histological grade, and Ki67 (All p-values ≥ 0.05). Furthermore, there was no significant association between PD-L1 expression with overall survival (OS) and disease-free survival (DFS). In assessment of TILs and survival relationship, the results showed that a high level of TILs was associated with long-term OS (hazard ratios (HR) = 0.48, 95% CI: 0.30 to 0.77, p-value < 0.001) and DFS (HR = 0.53, 95% CI: 0.35 to 0.78, p-value < 0.001). The results displayed that tumoral PD-L1 expression was strongly associated with high levels of TILs in TNBC patients (OR = 8.34, 95% CI: 2.68 to 25.95, p-value < 0.001). In conclusion, the study has shown the prognostic value of TILs and a strong association between tumoral PD-L1 overexpression with TILs in TNBC patients. [ABSTRACT FROM AUTHOR]

Simple Summary: The environment surrounding primary glioma brain tumors plays a critical role in tumor development and progression. Immune cells are important constituents of this tumor microenvironment. Classically, immune activation is thought to halt tumor progression; however, recent evidence has shown that glioma cells can interact with immune cells to convert them into tumor-supporting cells. These reprogrammed immune cells allow glioma cells to evade the anti-tumor effects of the immune system and can even promote tumor growth. Understanding the mechanisms that result in this altered immune tumor microenvironment may pave the way to new therapies for brain tumors. This review aims to provide an updated view of the mechanisms underlying glioma–immune cell interactions and novel therapies being developed to combat glioma immune evasion. Glioma progression is a complex process controlled by molecular factors that coordinate the crosstalk between tumor cells and components of the tumor microenvironment (TME). Among these, immune cells play a critical role in cancer survival and progression. The complex interplay between cancer cells and the immune TME influences the outcome of immunotherapy and other anti-cancer therapies. Here, we present an updated view of the pro- and anti-tumor activities of the main myeloid and lymphocyte cell populations in the glioma TME. We review the underlying mechanisms involved in crosstalk between cancer cells and immune cells that enable gliomas to evade the immune system and co-opt these cells for tumor growth. Lastly, we discuss the current and experimental therapeutic options being developed to revert the immunosuppressive activity of the glioma TME. Knowledge of the complex interplay that elapses between tumor and immune cells may help develop new combination treatments able to overcome tumor immune evasion mechanisms and enhance response to immunotherapies. [ABSTRACT FROM AUTHOR]

Trabectedin (TRB) and Lurbinectedin (LUR) are alkaloid compounds originally isolated from Ecteinascidia turbinata with proven antitumoral activity. Both molecules are structural analogues that differ on the tetrahydroisoquinoline moiety of the C subunit in TRB, which is replaced by a tetrahydro-β-carboline in LUR. TRB is indicated for patients with relapsed ovarian cancer in combination with pegylated liposomal doxorubicin, as well as for advanced soft tissue sarcoma in adults in monotherapy. LUR was approved by the FDA in 2020 to treat metastatic small cell lung cancer. Herein, we systematically summarise the origin and structure of TRB and LUR, as well as the molecular mechanisms that they trigger to induce cell death in tumoral cells and supporting stroma cells of the tumoral microenvironment, and how these compounds regulate immune cell function and fate. Finally, the novel therapeutic venues that are currently under exploration, in combination with a plethora of different immunotherapeutic strategies or specific molecular-targeted inhibitors, are reviewed, with particular emphasis on the usage of immune checkpoint inhibitors, or other bioactive molecules that have shown synergistic effects in terms of tumour regression and ablation. These approaches intend to tackle the complexity of managing cancer patients in the context of precision medicine and the application of tailor-made strategies aiming at the reduction of undesired side effects. [ABSTRACT FROM AUTHOR]

Simple Summary: The major histocompatibility complex type II is downregulated in glioblastoma (GB) due to the silencing of the major transcriptional regulator class II transactivator (CIITA). Recent studies have posed CIITA as a possible target in treating patients with GB. However, our results show that in mice, vaccination experiments with wildtype (-WT) and CIITA-expressing cells (-CIITA) equivalently protect against subsequent intracerebral challenges with GL261 cells. Adoptive cell transfer experiments likewise showed a similar antitumor potential of lymphocytes harvested from mice implanted intracerebrally with GL261-WT or -CIITA. Human GB-infiltrating myeloid cells, lymphocytes and NK cells remained mostly inactivated and/or tumor-supportive when in co-culture with GB cells, regardless of CIITA. Altogether, these results question the therapeutic potential of CIITA-mediated immunotherapy in glioblastoma. Background: The major histocompatibility complex type II is downregulated in glioblastoma (GB) due to the silencing of the major transcriptional regulator class II transactivator (CIITA). We investigated the pro-immunogenic potential of CIITA overexpression in mouse and human GB. Methods: The intracerebral growth of wildtype GL261-WT cells was assessed following contralateral injection of GL261-CIITA cells or flank injections with GL261-WT or GL261-CIITA cells. Splenocytes obtained from mice implanted intracerebrally with GL261-WT, GL261-CIITA cells or phosphate buffered saline (PBS) were transferred to other mice and subsequently implanted intracerebrally with GL261-WT. Human GB cells and (syngeneic) GB-infiltrating immune cells were isolated from surgical samples and co-cultured with GB cells expressing CIITA or not, followed by RT-qPCR assessment of the expression of key immune regulators. Results: Intracerebral vaccination of GL261-CIITA significantly reduced the subsequent growth of GL261-WT cells implanted contralaterally. Vaccination with GL261-WT or -CIITA subcutaneously, however, equivalently retarded the intracerebral growth of GL261 cells. Adoptive cell transfer experiments showed a similar antitumor potential of lymphocytes harvested from mice implanted intracerebrally with GL261-WT or -CIITA. Human GB-infiltrating myeloid cells and lymphocytes were not activated when cultured with CIITA-expressing GB cells. Tumor-infiltrating NK cells remained mostly inactivated when in co-culture with GB cells, regardless of CIITA. Conclusion: these results question the therapeutic potential of CIITA-mediated immunotherapy in glioblastoma. [ABSTRACT FROM AUTHOR]

With the expansion of urban areas, the amount of sludge produced by sewage treatment plants is increasing, raising big problems regarding the reintroduction of this sludge into nature in order to fully solve the wastewater problem. The application of sludge to agricultural surfaces or degraded land is a controversial solution since, despite the well-known benefits, sludge can, in certain cases, represent a real threat to both human health and the environment, with long-term harmful effects. The present study evaluates the potential genotoxicity of sludge using the Comet Test and three cellular bioindicators (lymphocytes, coelomocytes, and Allium cepa L.) for its quantification. To perform the tests, the soluble fraction of the sludge was used at concentrations of 25%, 50%, 75%, and 100%, as well as a negative control (H2O) and a positive control (H2O2). The Comet test indicated an increase in DNA damage among cells exposed for 4 h in the following order: coelomocytes, lymphocytes, and Allium cepa L. cells. Our results indicate that Allium cepa L. nuclei are more sensitive, with genotoxic effects being evident at concentrations as low as 25%. In coelomocytes, we recorded nuclear damage starting at a concentration of 75%. These results indicate the necessity of using multiple genotoxicity tests, combined in a test battery, to achieve a greater level of relevance. The concentration of the soluble fraction of the sludge has an inverse relationship with the auxin content in leaves and roots, suggesting varying levels of stress. The results of this study can contribute to the creation of a genotoxic profile of sewage sludge, facilitating decisions related to reducing its negative impact. [ABSTRACT FROM AUTHOR]

Simple Summary: Gastric cancer (GC) has a poor prognosis, as it has often grown into an advanced stage when diagnosed. Genetic testing is crucial for establishing a treatment plan. In this study, next-generation sequencing (NGS) was performed to discover that LRP1B mutation was associated with a poor prognosis in GC. Through layer-by-layer research, it was found that LRP1B expression in GC was accompanied by a higher-level infiltration of CD4+ T, CD8+ T cells, and CD86/CD163. It is concluded that LRP1B expression can stimulate tumor immune cell infiltration, thus bringing clinical benefits to GC patients. Background: Recent studies have shown that low-density lipoprotein receptor-related protein 1b (LRP1B), as a potential tumor suppressor, is implicated in the response to immunotherapy. The frequency of LRP1B mutation gene is high in many cancers, but its role in gastric cancer (GC) has not been determined. Methods: The prognostic value of LRP1B mutation in a cohort containing 100 patients having received radical gastrectomy for stage II–III GC was explored. By analyzing the data of LRP1B mRNA, the risk score of differentially expressed genes (DEGs) between LRP1B mutation-type and wild-type was constructed based on the TCGA-STAD cohort. The infiltration of tumor immune cells was evaluated by the CYBERSORT algorithm and verified by immunohistochemistry. Results: LRP1B gene mutation was an independent risk factor for disease-free survival (DFS) in GC patients (HR = 2.57, 95% CI: 1.28–5.14, p = 0.008). The Kaplan–Meier curve demonstrated a shorter survival time in high-risk patients stratified according to risk score (p < 0.0001). CYBERSORT analysis showed that the DEGs were mainly concentrated in CD4+ T cells and macrophages. TIMER analysis suggested that LRP1B expression was associated with the infiltration of CD4+ T cells and macrophages. Immunohistochemistry demonstrated that LRP1B was expressed in the tumor cells (TCs) and immune cells in 16/89 and 26/89 of the cohort, respectively. LRP1B-positive TCs were associated with higher levels of CD4+ T cells, CD8+ T cells, and CD86/CD163 (p < 0.05). Multivariate analysis showed that LRP1B-positive TCs represented an independent protective factor of DFS in GC patients (HR = 0.43, 95% CI: 0.10–0.93, p = 0.042). Conclusions: LRP1B has a high prognostic value in GC. LRP1B may stimulate tumor immune cell infiltration to provide GC patients with survival benefits. [ABSTRACT FROM AUTHOR]

Multiple system atrophy (MSA) is generally a sporadic neurodegenerative disease which ranks among atypical Parkinson's syndromes. The main clinical manifestation is a combination of autonomic dysfunction and parkinsonism and/or cerebellar disability. The disease may resemble other Parkinsonian syndromes, such as Parkinson's disease (PD) or progressive supranuclear palsy (PSP), from which MSA could be hardly distinguishable during the first years of progression. Due to the lack of a reliable and easily accessible biomarker, the diagnosis is still based primarily on the clinical picture. Recently, reduced levels of coenzyme Q10 (CoQ10) were described in MSA in various tissues, including the central nervous system. The aim of our study was to verify whether the level of CoQ10 in plasma and lymphocytes could serve as an easily available diagnostic biomarker of MSA. The study reported significantly lower levels of CoQ10 in the lymphocytes of patients with MSA compared to patients with PD and controls. The reduction in CoQ10 levels in lymphocytes correlated with the increasing degree of clinical involvement of patients with MSA. CoQ10 levels in lymphocytes seem to be a potential biomarker of disease progression. [ABSTRACT FROM AUTHOR]

We intended to investigate the presence and medical application of serum hypoxia-inducible factor-1 alpha (HIF-1α) along with the already known systemic inflammatory markers and the new one's inflammatory indices, the proportion of mean corpuscular volume and lymphocytes (MCVL) and the cumulative inflammatory index (IIC), for patients with ulcerative colitis (UC). We sought to establish correlations that may be present between the serum levels of HIF-1α and these inflammatory indices, as well as their relationship with disease activity and the extent of UC, which can provide us with a more precise understanding of the evolution, prognosis, and future well-being of patients. Serum samples were collected from 46 patients diagnosed with UC and 23 controls. For our assessment of the serum levels of HIF-1α, we used the Enzyme-Linked Immunosorbent Assay (ELISA) technique. Thus, for HIF-1α we detected significantly higher values in more severe and more extensive UC. When it came to MCVL and IIC, we observed statistically significant differences between the three groups being compared (Severe, Moderate, and Mild). Our study highlighted that HIF-1α correlated much better with a disease activity score, MCVL, and IIC. With MCVL and IIC, a strong and very strong correlation had formed between them and well-known inflammation indices. By examining the ROC curves of the analyzed parameters, we recognized that TWI (accuracy of 83.70%) provides the best discrimination of patients with early forms of UC, followed by HIF-1α (73.90% accuracy), MCVL (70.90% accuracy), and PLR (70.40%). In our study, we observed that HIF-1α, MCVL, and PLR had the same sensitivity (73.33%) but HIF-1α had a much better specificity (60.87% vs. 58.70%, and 54.35%). Also, in addition to the PLR, HIF-1α and MCVL can be used as independent predictor factors in the discrimination of patients with early forms of UC. [ABSTRACT FROM AUTHOR]

Lymphoepithelioma-like carcinoma of the skin (LELCS) is a rare primary skin cancer, with an annual incidence of 1/100,000 and about 85 cases published in the literature. It is considered the cutaneous counterpart of undifferentiated nasopharyngeal carcinoma (UNC, Schmincke–Regaud tumor) but has no association with EBV. We present an interesting case with features of LELCS in a 93-year-old man, right frontal–orbital region, diagnosed histologically and with immunohistochemical features. We also emphasize contrasting morphologic features for correct nosographic classification and address current issues, suggesting potential insights. Finally, we briefly reviewed other cases described in the literature. [ABSTRACT FROM AUTHOR]

Simple Summary: Our research explores the realm of immune cells to better understand patients with chronic lymphocytic leukemia (CLL) and common variable immunodeficiency (CVID). By examining immune checkpoints such as PD-1/PD-L1, CTLA-4/CD86, and CD200R/CD200 in different blood lymphocyte subpopulations, we aim to extract valuable insights that may improve not only our understanding of these diseases but also contribute to improving treatment conditions and the occurrence of subsequent complications and the development of hematological cancers. We hope that the knowledge about the role of these signaling pathways in the development and progression of these two diseases will allow us to develop modern and personalized diagnostic and therapeutic strategies, which in the future may allow monitoring the immune system of patients with CVID and CLL. This study aims to gain a deeper understanding of chronic lymphocytic leukemia (CLL) and common variable immunodeficiency (CVID) by studying immune cells and specific immune checkpoint signaling pathways. The analysis of the percentage of selected immune points and their ligands (PD-1/PD-L1, CTLA-4/CD86, and CD200R/CD200) on peripheral blood lymphocyte subpopulations was performed using flow cytometry, and additional analyses determining the serum concentration of the above-mentioned molecules were performed using enzyme immunoassay tests. The obtained results indicate several significant changes in the percentage of almost all tested molecules on selected subpopulations of T and B lymphocytes in both CVID and CLL patients in relation to healthy volunteers and between the disease subunits themselves. The results obtained were also supported by the analysis of the serum concentration of soluble molecules tested. By uncovering valuable insights, we hope to enhance our comprehension and management of these conditions, considering both immunodeficiencies and hematological malignancies. Understanding the role of these signaling pathways in disease development and progression may lead to the development of modern, personalized diagnostic and therapeutic strategies. Ultimately, this knowledge may enable the monitoring of the immune system in patients with CVID and CLL, paving the way for improved patient care in the future. [ABSTRACT FROM AUTHOR]

New molecular predictors for the response to treatment in HBV (hepatitis B virus) infection are assessed. Among them is miR-122. Our article searches the connection between miR-122 and the counts of lymphocytes in chronic HBV patients receiving treatment. We included the sera of 38 Romanian subjects with chronic HBV infection (20 receiving treatment and 18 not receiving treatment) and 5 healthy controls. The expression of miR-122 was determined using RT-PCR (real-time PCR) and a 2−ΔΔCT method. Two systematic analyses were also performed on databases (PUBMED, Web of Science, and Science Direct), eliminating systematic reviews, editorials, letters to editors, meta-analyses, reviews, conference proceedings, or pre-print manuscripts. We included human-based articles following the PRISMA criteria and the Newcastle Ottawa Assessment Scale for Case–Control and Cohort studies. R 4.2.2 was used for statistics, and MIENTURNET and STRING were used for the bioinformatic analysis. Our results showed a link between the variations in the expression of miR-122 and the counts of lymphocytes in HBV Romanian patients receiving therapy. Treatment influenced miR-122 and the lymphocyte numbers. This is the first study with these results, and it may lead to a new perspective on the inter-relationships between microRNAs and therapy in HBV patients. [ABSTRACT FROM AUTHOR]

Atherosclerosis is a chronic progressive disease that involves damage to the intima, inflammatory cell recruitment and the accumulation of lipids followed by calcification and plaque rupture. Inflammation is considered a key mediator of many events during the development and progression of the disease. Various types of inflammatory cells are reported to be involved in atherosclerosis. In the present paper, we discuss the involved inflammatory cells, their characteristic and functional significance in the development and progression of atherosclerosis. The detailed understanding of the role of all these cells in disease progression at different stages sheds more light on the subject and provides valuable insights as to where and when therapy should be targeted. [ABSTRACT FROM AUTHOR]

Simple Summary: Lymphoma is a heterogeneous group of neoplasms including over 70 different subtypes. Its biological characteristic of deriving from lymphoid tissues makes it ideal for immunotherapy. In this paper, we provide insights into lymphoma-specific clinical trials based on cytokine-induced killer (CIK) cell therapy. We also reviewed pre-clinical lymphoma models where CIK cells have been used along with other synergetic tumor-targeting immune modules to improve their therapeutic potential. From a broader perspective, we will highlight that CIK cell therapy has potential, and in this rapidly evolving landscape of cancer therapies its optimization (as a personalized therapeutic approach) will be beneficial in lymphomas. Cancer is a complex disease where resistance to therapies and relapses often pose a serious clinical challenge. The scenario is even more complicated when the cancer type itself is heterogeneous in nature, e.g., lymphoma, a cancer of the lymphocytes which constitutes more than 70 different subtypes. Indeed, the treatment options continue to expand in lymphomas. Herein, we provide insights into lymphoma-specific clinical trials based on cytokine-induced killer (CIK) cell therapy and other pre-clinical lymphoma models where CIK cells have been used along with other synergetic tumor-targeting immune modules to improve their therapeutic potential. From a broader perspective, we will highlight that CIK cell therapy has potential, and in this rapidly evolving landscape of cancer therapies its optimization (as a personalized therapeutic approach) will be beneficial in lymphomas. [ABSTRACT FROM AUTHOR]

TCR-like chimeric antigen receptor (CAR-T) cell therapy has emerged as a game-changing strategy in cancer immunotherapy, offering a broad spectrum of potential antigen targets, particularly in solid tumors containing intracellular antigens. In this study, we investigated the cytotoxicity and functional attributes of in vitro-generated T-lymphocytes, engineered with a TCR-like CAR receptor precisely targeting the cancer testis antigen MAGE-A4. Through viral transduction, T-cells were genetically modified to express the TCR-like CAR receptor and co-cultured with MAGE-A4-expressing tumor cells. Flow cytometry analysis revealed a significant surge in cells expressing activation markers CD69, CD107a, and FasL upon encountering tumor cells, indicating robust T-cell activation and cytotoxicity. Moreover, immune transcriptome profiling unveiled heightened expression of pivotal T-effector genes involved in immune response and cell proliferation regulation. Additionally, multiplex assays also revealed increased cytokine production and cytotoxicity driven by granzymes and soluble Fas ligand (sFasL), suggesting enhanced anti-tumor immune responses. Preliminary in vivo investigations revealed a significant deceleration in tumor growth, highlighting the therapeutic potential of these TCR-like CAR-T cells. Further investigations are warranted to validate these revelations fully and harness the complete potential of TCR-like CAR-T cells in overcoming cancer's resilient defenses. [ABSTRACT FROM AUTHOR]

Background and Aim: Immune status profile can predict response to vaccination, while lymphocyte phenotypic alterations represent its effectiveness. We prospectively evaluated these parameters in kidney transplant recipients (KTRs) regarding Tozinameran (BNT162b2) vaccination. Method: In this prospective monocenter observational study, 39 adult KTRs, on stable immunosuppression, naïve to COVID-19, with no protective humoral response after two Tozinameran doses, received the third vaccination dose, and, based on their immunity activation, they were classified as responders or non-responders. Humoral and cellular immunities were assessed at predefined time points (T0: 48 h before the first, T1: 48 h prior to the third and T2: three weeks after the third dose). Results: Responders, compared to non-responders, had a higher total and transitional B-lymphocyte count at baseline (96.5 (93) vs. 51 (52)cells/μL, p: 0.045 and 9 (17) vs. 1 (2)cells/μL, p: 0.031, respectively). In the responder group, there was a significant increase, from T0 to T1, in the concentrations of activated CD4+ (from 6.5 (4) to 10.08 (11)cells/μL, p: 0.001) and CD8+ (from 8 (19) to 14.76 (16)cells/μL, p: 0.004) and a drop in CD3+PD1+ T-cells (from 130 (121) to 30.44 (25)cells/μL, p: 0.001), while naïve and transitional B-cells increased from T1 to T2 (from 57.55 (66) to 1149.3 (680)cells/μL, p < 0.001 and from 1.4 (3) to 17.5 (21)cells/μL, p: 0.003). The percentages of memory and marginal zone B-lymphocytes, and activated CD4+, CD8+ and natural killer (NK) T-cells significantly increased, while those of naïve B-cells and CD3+PD1+ T-cells reduced from T0 to T1. Conclusions: Responders and non-responders to the third BNT162b2 dose demonstrated distinct initial immune cell profiles and changes in cellular subpopulation composition following vaccination. [ABSTRACT FROM AUTHOR]

Adoptive T-cell therapies tailored for the treatment of solid tumors encounter intricate challenges, necessitating the meticulous selection of specific target antigens and the engineering of highly specific T-cell receptors (TCRs). This study delves into the cytotoxicity and functional characteristics of in vitro-cultured T-lymphocytes, equipped with a TCR designed to precisely target the cancer-testis antigen NY-ESO-1. Flow cytometry analysis unveiled a notable increase in the population of cells expressing activation markers upon encountering the NY-ESO-1-positive tumor cell line, SK-Mel-37. Employing the NanoString platform, immune transcriptome profiling revealed the upregulation of genes enriched in Gene Ontology Biological Processes associated with the IFN-γ signaling pathway, regulation of T-cell activation, and proliferation. Furthermore, the modified T cells exhibited robust cytotoxicity in an antigen-dependent manner, as confirmed by the LDH assay results. Multiplex immunoassays, including LEGENDplex™, additionally demonstrated the elevated production of cytotoxicity-associated cytokines driven by granzymes and soluble Fas ligand (sFasL). Our findings underscore the specific targeting potential of engineered TCR T cells against NY-ESO-1-positive tumors. Further comprehensive in vivo investigations are essential to thoroughly validate these results and effectively harness the intrinsic potential of genetically engineered T cells for combating cancer. [ABSTRACT FROM AUTHOR]

The mechanisms underlying the immune response to coronavirus disease 2019 (COVID-19) and the recovery process have not been fully elucidated. The aim of the study was to analyze leukocyte subpopulations in patients at significant time points (at diagnosis, and 3 and 6 months after infection) selected according to the analysis of changes in the lungs by the CT classification system, considering the severity of the disease. The study groups consisted of severe and non-severe COVID-19 patients. There was a significant decrease in CD8+ T cells, NK and eosinophils, with an increasing percentage of neutrophils during hospitalization. We noticed lower levels of CD4 and CD8 T lymphocytes, eosinophils, basophils, and CD16+ monocytes and elevated neutrophil levels in severe patients relative to non-severe patients. Three months after infection, we observed higher levels of basophils, and after 6 months, higher CD4/CD8 ratios and T cell levels in the severe compared to non-severe group. Non-severe patients showed significant changes in the leukocyte populations studied at time of hospitalization and both within 3 months and 6 months of onset. The CT CSS classification with parameters of the flow cytometry method used for COVID-19 patients determined changes that proved useful in the initial evaluation of patients. [ABSTRACT FROM AUTHOR]

Simple Summary: According to guidelines, resection remains a gold standard treatment in early-stage NSCLC. Because of the curative potential of surgery in these patients, microscopical and microenvironmental tumor processes in localized (N0) disease have been superseded for a long time and is a new emerging research field. Here, we investigated the influence of pathological variables and tumor immune environment in terms of survival and recurrence in resected adenocarcinomas staged I-IIA. Microscopical predictors and Tumor Immune Microenvironment (TIME) have been studied less in early-stage NSCLC due to the curative intent of resection and the satisfactory survival rate achievable. Despite this, the emerging literature enforces the role of the immune system and microscopical predictors as prognostic variables in NSCLC and in adenocarcinomas (ADCs) as well. Here, we investigated whether cancer-related microscopical variables and TIME influence survival and recurrence in I-IIA ADCs. We retrospectively collected I-IIA ADCs treated (lobectomy or segmentectomy) at the University Hospital (Padova) between 2016 and 2022. We assigned to pathological variables a cumulative pathological score (PS) resulting as the sum of them. TIME was investigated as tumor-infiltrating lymphocytes (TILs < 11% or ≥11%) and PD-L1 considering its expression (<1% or ≥1%). Then, we compared survival and recurrence according to PS, histology, TILs and PD-L1. A total of 358 I-IIA ADCs met the inclusion criteria. The median PS grew from IA1 to IIA, indicating an increasing microscopical cancer activity. Except for the T-SUVmax, any pathological predictor seemed to be different between PD-L1 < 1% and ≥1%. Histology, PS, TILs and PD-L1 were unable to indicate a survival difference according to the Log-rank test (p = 0.37, p = 0.25, p = 0.41 and p = 0.23). Even the recurrence was non-significant (p = 0.90, p = 0.62, p = 0.97, p = 0.74). According to our findings, resection remains the best upfront treatment in I-IIA ADCs. Microscopical cancer activity grows from IA1 to IIA tumors, but it does not affect outcomes. These outcomes are also unmodified by TIME. Probably, microscopical cancer development and immune reaction against cancer are overwhelmed by an adequate R0-N0 resection. [ABSTRACT FROM AUTHOR]

Simple Summary: Triple-negative breast cancer (TNBC) is generally malignant and has a poor prognosis. New biomarkers and therapeutic strategies are therefore needed. In this study, we investigated whether granzyme B (GZMB) in the tumor microenvironment can be a biomarker of therapeutic response and prognosis in TNBC via the immunohistochemical staining of clinical specimens from 230 patients with primary TNBC. The key results were in the programmed cell death ligand 1 (PD-L1)-positive group, where GZMB-high TNBC patients had better recurrence-free survival and overall survival than GZMB-low patients. A multivariate analysis also showed significantly better overall survival in PD-L1-positive and GZMB-high patients. These results indicate that GZMB is a useful prognostic marker in PD-L1-positive TNBC patients. Tumor-infiltrating lymphocytes in the tumor microenvironment are important in the treatment of triple-negative breast cancer (TNBC). Cytotoxic T cells produce cytokines and cytotoxic factors, such as perforin and granzyme, which induce apoptosis by damaging target cells. To identify biomarkers of these cells, we investigated granzyme B (GZMB) in the tumor microenvironment as a biomarker of treatment response and prognosis in 230 patients with primary TNBC who underwent surgery without preoperative chemotherapy between January 2004 and December 2014. Programmed cell death ligand 1 (PD-L1) positivity was defined as a composite positive score ≥10 based on the PD-L1 immunostaining of tumor cells and immune cells. GZMB-high was defined as positivity in ≥1% of tumor-infiltrating lymphocytes (TILs). Among the 230 TNBC patients, 117 (50.9%) had CD8-positive infiltrating tumors. In the PD-L1-positive group, a Kaplan–Meier analysis showed that GZMB-high TNBC patients had better recurrence-free survival (RFS) and overall survival (OS) than GZMB-low patients and that OS was significantly longer (RFS: p = 0.0220, OS: p = 0.0254). A multivariate analysis also showed significantly better OS in PD-L1- and GZMB-high patients (hazard ratio: 0.25 (95% IC: 0.07–0.88), p = 0.03). Our findings indicate that GZMB is a useful prognostic biomarker in PD-L1-positive TNBC patients. [ABSTRACT FROM AUTHOR]

5-methylcytosine (5mC) is one of the most important epigenetic modifications. Its increased occurrence in regulatory sequences of genes, such as promoters and enhancers, is associated with the inhibition of their expression. Methylation patterns are not stable but are sensitive to factors such as the environment, diet, and age. In the present study, we investigated the effects of fungicide miconazole, both alone and in combination with the insecticide Mospilan 20SP, on the methylation status of bovine GSTP1, GSTA4, and AChE genes in bovine lymphocytes cultured in vitro. The methylation-specific PCR technique was used for the objectives of this study. We found that miconazole alone at concentrations of 1.25, 2.5, 5, 10, 25, and 50 µg/mL after 24 h exposure probably did not induce changes in methylation for all three genes analysed. The same results were found for the combination of pesticides at 24 h exposure and the following concentrations for each of them: 0.625, 1.25, 2.5, 5, and 12.5 µg/mL. Thus, we can conclude that the fungicide miconazole alone, as well as in combination with the insecticide Mospilan 20SP, was unlikely to cause changes to the methylation of bovine GSTP1, GSTA4, and AChE genes. [ABSTRACT FROM AUTHOR]

Lymphocytes are key players in the pathogenesis of multiple sclerosis and a distinct target of several immunomodulatory treatment strategies. In this study, we aim to evaluate the effect of various pre-analytic conditions on immune cell counts to conclude the relevance for clinical implications. Twenty healthy donors were assessed for the effects of distinct storage temperatures and times after blood draws, different durations of tourniquet application, body positions and varying aspiration forces during blood draws. Immune cell frequencies were analyzed using multicolor flowcytometry. While storage for 24 h at 37 °C after blood draws was associated with significantly lower cell counts, different durations of tourniquet application, body positions and varying aspirations speeds did not have significant impacts on the immune cell counts. Our data suggest that immune cell counts are differently affected by pre-analytic conditions being more sensitive to storage temperature. Pre-analytic conditions should be carefully considered when interpreting the laboratory values of immune cell subpopulations. [ABSTRACT FROM AUTHOR]

The mortality of COVID-19 patients has left the world devastated. Many scoring systems have been developed to predict the mortality of COVID-19 patients, but several scoring components cannot be carried out in limited health facilities. Herein, the authors attempted to create a new and easy scoring system involving mean arterial pressure (MAP), PF Ratio, or SF ratio-respiration rate (SF Ratio-R), and lymphocyte absolute, which were abbreviated as MPL or MSLR functioning, as a predictive scoring system for mortality within 30 days for COVID-19 patients. Of 132 patients with COVID-19 hospitalized between March and November 2021, we followed up on 96 patients. We present bivariate and multivariate analyses as well as the area under the curve (AUC) and Kaplan–Meier charts. From 96 patients, we obtained an MPL score of 3 points: MAP < 75 mmHg, PF Ratio < 200, and lymphocyte absolute < 1500/µL, whereas the MSLR score was 6 points: MAP < 75 mmHg, SF Ratio < 200, lymphocyte absolute < 1500/µL, and respiration rate 24/min. The MPL cut-off point is 2, while the MSLR is 4. MPL and MSLR have the same sensitivity (79.1%) and specificity (75.5%). The AUC value of MPL vs. MSLR was 0.802 vs. 0.807. The MPL ≥ 2 and MSLR ≥ 4 revealed similar predictions for survival within 30 days (p < 0.05). Conclusion: MPL and MSLR scores are potential predictors of mortality in COVID-19 patients within 30 days in a resource-limited country. [ABSTRACT FROM AUTHOR]

Simple Summary: Single-cell RNA sequencing has significantly contributed to the discovery of heterogenous cellular programs that are highly expressed by distinct cell subtypes. Yet, the quantification of cell subtype-specific and shared gene co-expressing modules (GEMs) associated with cell differentiation is a challenge. Herein, we have developed scGEM to uncover such hidden GEMs and conducted a systematic evaluation of model performance as well as a comparison with existing methods. We demonstrate that scGEM has the potential to generate a better biological explanation of GEMs using simulated and real-world datasets. The positive impacts of this study can illuminate the interpretation of gene modules in single-cell transcriptome analysis and shed light on the cell–cell communication and regulatory network. Background: Single-cell transcriptome analysis has fundamentally changed biological research by allowing higher-resolution computational analysis of individual cells and subsets of cell types. However, few methods have met the need to recognize and quantify the underlying cellular programs that determine the specialization and differentiation of the cell types. Methods: In this study, we present scGEM, a nested tree-structured nonparametric Bayesian model, to reveal the gene co-expression modules (GEMs) reflecting transcriptome processes in single cells. Results: We show that scGEM can discover shared and specialized transcriptome signals across different cell types using peripheral blood mononuclear single cells and early brain development single cells. scGEM outperformed other methods in perplexity and topic coherence (p < 0.001) on our simulation data. Larger datasets, deeper trees and pre-trained models are shown to be positively associated with better scGEM performance. The GEMs obtained from triple-negative breast cancer single cells exhibited better correlations with lymphocyte infiltration (p = 0.009) and the cell cycle (p < 0.001) than other methods in additional validation on the bulk RNAseq dataset. Conclusions: Altogether, we demonstrate that scGEM can be used to model the hidden cellular functions of single cells, thereby unveiling the specialization and generalization of transcriptomic programs across different types of cells. [ABSTRACT FROM AUTHOR]

The assessment of the nutritional and inflammatory status of paediatric patients with coeliac disease is an interesting approach to early diagnosis and functional follow-up. Most authors agree that the normalisation of symptoms takes about one year. The aim of the study was to evaluate the clinical manifestation and normalisation of routine analytics in Spanish children diagnosed with celiac disease. Methods: We performed a retrospective case–control study in Spanish paediatric patients, including 21 celiac patients and 20 healthy controls. The 21 patients selected in the case–control study were followed for 5 years after starting a gluten-free diet (GFD). All patients had type 3 villous atrophy according to the Marsh–Oberhuber classification. A total of 39 blood samples were taken before the start of the GFD, and 109 were taken after. Twenty control sera from healthy donors were used for comparison. Results: We found that patients had a subclinical but statistically significant increase in blood calcium, transaminases, and white blood cells, and a decrease in serum iron, at the time of diagnosis. Our study also shows that analytical values normalise within five years on a gluten-free diet. Conclusions: The use of a combination of subclinical changes, including low iron, high calcium, elevated leukocytes, lymphocytes, and ALT levels in blood samples, together with a low growth percentile, is pertinent in detecting coeliac disease. This set of parameters could help in the diagnosis of patients without clinical symptoms. We can also show that the levels of Fe, Ca, transaminases, and leucocytes remain subclinically altered after 3 years, despite the gluten-free diet. [ABSTRACT FROM AUTHOR]

TGF-β signaling promotes migration, invasion, and distant colonization of cancer cells in advanced metastatic cancers. TGF-β signaling suppresses the anti-tumor immune response in a tumor microenvironment, allowing sustained tumor growth. TGF-β plays an important role in normal physiology; thus it is no surprise that the clinical development of effective and safe TGF-β inhibitors has been hampered due to their high toxicity. We discovered that increased expression of LY6K in cancer cells led to increased TGF-β signaling and that inhibition of LY6K could lead to reduced TGF-β signaling and reduced in vivo tumor growth. LY6K is a highly cancer-specific protein, and it is not expressed in normal organs except in the testes. Thus, LY6K is a valid target for developing therapeutic strategies to inhibit TGF-β signaling in cancer cells. We employed in vitro pull-down assays and molecular dynamics simulations to understand the structural determinants of the TGF-β receptor complex with LY6K. This combined approach allowed us to identify the critical residues and dynamics of the LY6K interaction with the TGF-β receptor complex. These data are critical in designing novel drugs for the inhibition of TGF-β in LY6K expressing cancer, induction of anti-tumor immune response, and inhibition of tumor growth and metastatic spread. [ABSTRACT FROM AUTHOR]

Bioactive mesoporous glass nanospheres (nanoMBGs) charged with antiosteoporotic drugs have great potential for the treatment of osteoporosis and fracture prevention. In this scenario, cells of the immune system are essential both in the development of disease and in their potential to stimulate therapeutic effects. In the present work, we hypothesize that nanoMBGs loaded with ipriflavone can exert a positive osteoimmune effect. With this objective, we assessed the effects of non-loaded and ipriflavone-loaded nanoparticles (nanoMBGs and nanoMBG-IPs, respectively) on CD4+ Th2 lymphocytes because this kind of cell is implicated in the inhibition of osseous loss by reducing the RANKL/OPG relationship through the secretion of cytokines. The results indicate that nanoMBGs enter efficiently in CD4+ Th2 lymphocytes, mainly through phagocytosis and clathrin-dependent mechanisms, without affecting the function of these T cells or inducing inflammatory mediators or oxidative stress, thus maintaining the reparative Th2 phenotype. Furthermore, the incorporation of the anti-osteoporotic drug ipriflavone reduces the potential unwanted inflammatory response by decreasing the presence of ROS and stimulating intracellular anti-inflammatory cytokine release like IL-4. These results evidenced that nanoMBG loaded with ipriflavone exerts a positive osteoimmune effect. [ABSTRACT FROM AUTHOR]

Bulgarian Rosa damascena Mill. is has been known since ancient times for its high-quality oil, hydrosol, and other aromatic products. Rose hydrosol has various biological activities, but no research on its anticytotoxic/antigenotoxic effects exists. This study aimed to assess its defense potential against the genotoxin N-methyl-N′-nitro-N-nitrosoguanidine and to test its cytotoxic/genotoxic activity in plant and human lymphocyte test systems. Endpoints for cytotoxicity (mitotic index and nuclear division index) and genotoxicity (chromosome aberration and micronuclei) were used. Hydrosol was applied as a single treatment in concentrations ranging from 3% to 20% (4 h) to assess its cytotoxic and genotoxic effects. Its protective potential against MNNG was tested by applying an experimental scheme involving (i) conditioning treatment with non-toxic or slightly toxic concentrations of hydrosol, followed by genotoxin challenge (50 μg/mL) with a 4 h intertreatment time and (ii) treatment with hydrosol and mutagen with no time between the treatments. Hydrosol induces low cytotoxicity and clastogenicity, demonstrating cytoprotective/genoprotective effects against the mutagen in both applied test systems. The hydrosol defense potential was expressed by a more than twofold reduction in both chromosomal aberrations and micronuclei and by enhancing the mitotic activity compared with that of the mutagen, regardless of the experimental conditions. The results are promising for further hydrosol applications in pharmaceutical and medical practice. [ABSTRACT FROM AUTHOR]

Simple Summary: The immune system consists of various mechanisms contributing to the battle against cancer cells or hazardous pathogens. However, in cancer progression the immune system is often unable to eliminate neoplastic cells, although immune effector cells infiltrate the tumor environment. The current paper reviews the causes for this immune escape. Specifically, we comprehensively discuss various roles of sialic acids in this process. Specific focus is given to adhesion molecules re-expressed on membranes of tumor cells, which carry oligo- and polysialic acid chains. These carrier proteins loaded with sialic acids direct the interaction between immune effector and tumor cells and thus prevent the "kiss of death" between the latter and the former cells. We also discuss strategies suited to reduce the degree of sialic acid presence on the surface of tumor cells, which can be the basis for future therapeutic intervention. Glycans linked to surface proteins are the most complex biological macromolecules that play an active role in various cellular mechanisms. This diversity is the basis of cell–cell interaction and communication, cell growth, cell migration, as well as co-stimulatory or inhibitory signaling. Our review describes the importance of neuraminic acid and its derivatives as recognition elements, which are located at the outermost positions of carbohydrate chains linked to specific glycoproteins or glycolipids. Tumor cells, especially from solid tumors, mask themselves by re-expression of hypersialylated neural cell adhesion molecule (NCAM), neuropilin-2 (NRP-2), or synaptic cell adhesion molecule 1 (SynCAM 1) in order to protect themselves against the cytotoxic attack of the also highly sialylated immune effector cells. More particularly, we focus on α-2,8-linked polysialic acid chains, which characterize carrier glycoproteins such as NCAM, NRP-2, or SynCam-1. This characteristic property correlates with an aggressive clinical phenotype and endows them with multiple roles in biological processes that underlie all steps of cancer progression, including regulation of cell–cell and/or cell–extracellular matrix interactions, as well as increased proliferation, migration, reduced apoptosis rate of tumor cells, angiogenesis, and metastasis. Specifically, re-expression of poly/oligo-sialylated adhesion molecules on the surface of tumor cells disrupts their interaction with immune-effector cells and contributes to pathophysiological immune escape. Further, sialylated glycoproteins induce immunoregulatory cytokines and growth factors through interactions with sialic acid-binding immunoglobulin-like lectins. We describe the processes, which modulate the interaction between sialylated carrier glycoproteins and their ligands, and illustrate that sialic acids could be targets of novel therapeutic strategies for treatment of cancer and immune diseases. [ABSTRACT FROM AUTHOR]

Simple Summary: A critical step in the success of immunotherapy is the presentation of tumor-derived peptides by the major histocompatibility complex I (MHC-I) of tumor cells. These neoantigens are potentially immunogenic and trigger immune responses orchestrated by cytotoxic cells. In malignant mesothelioma (MM), tumor development is nevertheless characterized by a low mutation rate despite major structural chromosomal rearrangements driving oncogenesis. In this paper, we propose a paradigm based on the mechanisms of the DNA damage tolerance (DDT) pathways to increase the frequency of non-synonymous mutations. The idea is to transiently activate the error-prone DDT in order to generate neoantigens while preserving a fully competent antitumor immune response. Immunotherapy based on two checkpoint inhibitors (ICI), programmed cell death 1 (PD-1, Nivolumab) and cytotoxic T-lymphocyte 4 (CTLA-4, Ipilimumab), has provided a significant improvement in overall survival for malignant mesothelioma (MM). Despite this major breakthrough, the median overall survival of patients treated with the two ICIs only reached 18.1 months vs. 14 months in standard chemotherapy. With an objective response rate of 40%, only a subset of patients benefits from immunotherapy. A critical step in the success of immunotherapy is the presentation of tumor-derived peptides by the major histocompatibility complex I (MHC-I) of tumor cells. These neoantigens are potentially immunogenic and trigger immune responses orchestrated by cytotoxic cells. In MM, tumor development is nevertheless characterized by a low mutation rate despite major structural chromosomal rearrangements driving oncogenesis (BAP1, NF2, CDKN2AB). In this opinion, we propose to investigate an approach based on the mechanisms of the DNA damage tolerance (DDT) pathways to increase the frequency of non-synonymous mutations. The idea is to transiently activate the error-prone DDT in order to generate neoantigens while preserving a fully competent antitumor immune response. [ABSTRACT FROM AUTHOR]

Titanium dental implants are one of the modalities to replace missing teeth. The release of titanium particles from the implant's surface may modulate the immune cells, resulting in implant failure. However, little is known about the immune microenvironment that plays a role in peri-implant inflammation as a consequence of titanium particles. In this study, the peri-implant gingival tissues were collected from patients with failed implants, successful implants and no implants, and then a whole transcriptome analysis was performed. The gene set enrichment analysis confirmed that macrophage M1/M2 polarization and lymphocyte proliferation were differentially expressed between the study groups. The functional clustering and pathway analysis of the differentially expressed genes between the failed implants and successful implants versus no implants revealed that the immune response pathways were the most common in both comparisons, implying the critical role of infiltrating immune cells in the peri-implant tissues. The H&E and IHC staining confirmed the presence of titanium particles and immune cells in the tissue samples, with an increase in the infiltration of lymphocytes and macrophages in the failed implant samples. The in vitro validation showed a significant increase in the level of IL-1β, IL-8 and IL-18 expression by macrophages. Our findings showed evidence that titanium particles modulate lymphocyte and macrophage polarization in peri-implant gingival tissues, which can help in the understanding of the imbalance in osteoblast–osteoclast activity and failure of dental implant osseointegration. [ABSTRACT FROM AUTHOR]

Simple Summary: Tumor-infiltrating lymphocytes have gained a critical role in the newly developed cancer immunotherapy methods. Traditionally, pathologists and researchers count and score these lymphocytes to evaluate the patient's response to the treatments. However, scoring the lymphocytes is conducted microscopically and highly depends on the pathologist and researcher's experiences. In this manuscript, we have proposed and developed an automated method, using a two-stage deep learning model to score the lymphocytes in breast cancer histopathology slides. We further verified the accuracy of our method by statistically comparing results from expert pathologists to the output from our developed model. Detection of tumor-infiltrating lymphocytes (TILs) in cancer images has gained significant importance as these lymphocytes can be used as a biomarker in cancer detection and treatment procedures. Our goal was to develop and apply a TILs detection tool that utilizes deep learning models, following two sequential steps. First, based on the guidelines from the International Immuno-Oncology Biomarker Working Group (IIOBWG) on Breast Cancer, we labeled 63 large pathology imaging slides and annotated the TILs in the stroma area to create the dataset required for model development. In the second step, various machine learning models were employed and trained to detect the stroma where U-Net deep learning structure was able to achieve 98% accuracy. After detecting the stroma area, a Mask R-CNN model was employed for the TILs detection task. The R-CNN model detected the TILs in various images and was used as the backbone analysis network for the GUI development of the TILs detection tool. This is the first study to combine two deep learning models for TILs detection at the cellular level in breast tumor histopathology slides. Our novel approach can be applied to scoring TILs in large cancer slides. Statistical analysis showed that the output of the implemented approach had 95% concordance with the scores assigned by the pathologists, with a p-value of 0.045 (n = 63). This demonstrated that the results from the developed software were statistically meaningful and highly accurate. The implemented approach in analyzing whole tumor histology slides and the newly developed TILs detection tool can be used for research purposes in biomedical and pathology applications and it can provide researchers and clinicians with the TIL score for various input images. Future research using additional breast cancer slides from various sources for further training and validation of the developed models is necessary for more inclusive, rigorous, and robust clinical applications. [ABSTRACT FROM AUTHOR]