ICOS-Positive Regulatory T Cells in Hepatocellular Carcinoma: The Perspective from Digital Pathology Analysis.

INTRODUCTION: Inducible co-stimulator (ICOS), an important co-stimulatory receptor on effector T cells (Teffs), may also contribute to tumor growth due to its high expression on regulatory T cells (Tregs). This study explored the clinical significance of ICOS-expressing Tregs in hepatocellular carcinoma (HCC). METHODS: Tumor tissues from HCC patients who received curative hepatectomy were obtained at a referral center. Dual immunohistochemistry was performed to evaluate the expression of ICOS and Foxp3. The cell densities and proximities between stained cells in regions of interest were measured by digital pathology and the associations with clinical outcome were analyzed. RESULTS: A total of 142 patients (male:female = 112: 30, median age of 61.0 years) were enrolled. Among them, 87 (61.3%) had chronic hepatitis B virus infection and 33 (23.2%) had chronic hepatitis C infection. Low α-fetoprotein level (<20 ng/mL) and early-stage were significantly associated with improved overall survival (OS). The density of ICOS+Foxp3+ cells and the ratio of ICOS+Foxp3+/total Foxp3+ cells were significantly higher (p < 0.001) in the tumor center than in the peritumor area. Patients with a high density of ICOS+Foxp3+ cells or a high ratio of ICOS+Foxp3+/total Foxp3+ cells in the tumor center trended to have a shorter OS. A shorter distance between ICOS+Foxp3+ cells and ICOS+Foxp3- cells (likely Teffs) in the tumor center was significantly associated with a shorter OS (p = 0.030), suggesting active immunosuppression of ICOS+ Tregs on ICOS+ Teffs. CONCLUSION: An increased abundance of ICOS+ Tregs in the tumor center in comparison to the peritumor area indicates a strong immunosuppressive tumor microenvironment of HCC. A high proportion of ICOS+Foxp3+ cells and a shorter distance between ICOS+ Tregs and other ICOS+ cells were associated with a poor OS, suggesting that depleting ICOS+ Tregs might provide clinical benefit for patients with HCC.

PDGF-AB and 5-Azacytidine induce conversion of somatic cells into tissue-regenerative multipotent stem cells.

Current approaches in tissue engineering are geared toward generating tissue-specific stem cells. Given the complexity and heterogeneity of tissues, this approach has its limitations. An alternate approach is to induce terminally differentiated cells to dedifferentiate into multipotent proliferative cells with the capacity to regenerate all components of a damaged tissue, a phenomenon used by salamanders to regenerate limbs. 5-Azacytidine (AZA) is a nucleoside analog that is used to treat preleukemic and leukemic blood disorders. AZA is also known to induce cell plasticity. We hypothesized that AZA-induced cell plasticity occurs via a transient multipotent cell state and that concomitant exposure to a receptive growth factor might result in the expansion of a plastic and proliferative population of cells. To this end, we treated lineage-committed cells with AZA and screened a number of different growth factors with known activity in mesenchyme-derived tissues. Here, we report that transient treatment with AZA in combination with platelet-derived growth factor-AB converts primary somatic cells into tissue-regenerative multipotent stem (iMS) cells. iMS cells possess a distinct transcriptome, are immunosuppressive, and demonstrate long-term self-renewal, serial clonogenicity, and multigerm layer differentiation potential. Importantly, unlike mesenchymal stem cells, iMS cells contribute directly to in vivo tissue regeneration in a context-dependent manner and, unlike embryonic or pluripotent stem cells, do not form teratomas. Taken together, this vector-free method of generating iMS cells from primary terminally differentiated cells has significant scope for application in tissue regeneration.

Human red blood cells release microvesicles with distinct sizes and protein composition that alter neutrophil phagocytosis.

Extracellular vesicles (EVs) are membrane-bound structures released by cells and tissues into biofluids, involved in cell-cell communication. In humans, circulating red blood cells (RBCs), represent the most common cell-type in the body, generating daily large numbers of microvesicles. In vitro, RBC vesiculation can be mimicked by stimulating RBCs with calcium ionophores, such as ionomycin and A23187. The fate of microvesicles released during in vivo aging of RBCs and their interactions with circulating cells is hitherto unknown. Using SEC plus DEG isolation methods, we have found that human RBCs generate microvesicles with two distinct sizes, densities, and protein composition, identified by flow cytometry, and MRPS, and further validated by immune TEM. Furthermore, proteomic analysis revealed that RBC-derived microvesicles (RBC-MVs) are enriched in proteins with important functions in ion channel regulation, calcium homeostasis, and vesicular transport, such as of sorcin, stomatin, annexin A7, and RAB proteins. Cryo-electron microscopy identified two separate pathways of RBC-MV-neutrophil interaction, direct fusion with the plasma membrane and internalization, respectively. Functionally, RBC-MVs decrease neutrophil ability to phagocytose E. coli but do not affect their survival at 24 hrs. This work brings new insights regarding the complexity of the RBC-MVs biogenesis, as well as their possible role in circulation.

Detailed bladder cancer immunoprofiling reveals new clues for immunotherapeutic strategies.

Objectives: Bladder cancer (BlCa) is the tenth most frequent malignancy worldwide and the costliest to treat and monitor. Muscle-invasive BlCa (MIBC) has a dismal prognosis, entailing the need for alternative therapies for the standard radical cystectomy. Checkpoint blockade immunotherapy has been approved for high-grade non-muscle-invasive BlCa (HG NMIBC) and metastatic disease, but its effectiveness in localised MIBC remains under scrutiny. Herein, we sought to characterise and compare the immune infiltrate of HG NMIBC and MIBC samples, including ICOS expression, a targetable immune checkpoint associated with regulatory T cell(Tregs)-mediated immunosuppression. Methods: Immunohistochemistry for CD83, CD20, CD68, CD163, CD3, CD8, CD4, FoxP3/ICOS and PD-L1 was performed in HG NMIBC and MIBC samples (n = 206), and positive staining was quantified in the peritumoral and/or intratumoral tissue compartments with QuPath imaging software. Results: CD20+ B cells, CD68+ and CD163+ tumor-associated macrophages were significantly increased in MIBCs and associated with poor prognosis. In turn, higher infiltration of T cells was associated with prolonged survival, with exception of the CD4+ helper subset. Intratumoral expression of CD3 and CD8 was independent prognostic factors for increased disease-free survival (DFS) in multivariable analysis. Remarkably, Tregs (FoxP3+/FoxP3+ICOS+) were found differentially distributed between tissue compartments. PD-L1 immunoexpression independently predicted a shorter DFS and associated with higher infiltration of FoxP3+ICOS+ Tregs. Conclusions: Immune infiltrates of HG NMIBC and MIBC display significant differences that may help selecting patients for immunotherapies. Considering ICOS immunoexpression results, it might constitute a relevant therapeutic target, eventually in combination with anti-PD-1/PD-L1 therapies, for certain BlCa patient subsets.

Effects of endurance racing on horse plasma extracellular particle miRNA.

BACKGROUND: Physical exercise is an essential factor in preventing and treating metabolic diseases by promoting systemic benefits throughout the body. The molecular factors involved in this process are poorly understood. Micro RNAs (miRNAs) are small non-coding RNAs that inhibit mRNA transcription. MiRNAs, which can participate in the benefits of exercise to health, circulate in plasma in extracellular particles (EP). Horses that undergo endurance racing are an excellent model to study the impact of long-duration/low intensity exercise in plasma EP miRNAs. OBJECTIVES: To evaluate the effects of 160 km endurance racing on horse plasma extracellular particles and their miRNA population. STUDY DESIGN: Cohort study. METHODS: We collected plasma from five Arabian horses during five time-points of an endurance ride. Extracellular particles were purified from plasma and characterised by electron microscopy, resistive pulse sensing (qNano) and western blotting. Small RNAs were purified from horse plasma EP, and sequencing was performed. RESULTS: Endurance racing increased EP concentration and average diameter compared to before the race. Western blotting showed a high concentration of extracellular vesicles proteins 2 hours after the race, which returned to baseline 15 hours after the race. MicroRNA differential expression analysis revealed increasing levels of eca-miR-486-5p during and after the race, and decreasing levels of eca-miR-9083 after the end. CONCLUSIONS: This study adds new data about the variation in plasma EP concentrations after long-distance exercise and brings new insights about the roles of exercise-derived EP miRNAs during low-intensity endurance exercise.

Simplicity DiffExpress: A Bespoke Cloud-Based Interface for RNA-seq Differential Expression Modeling and Analysis.

One of the key challenges for transcriptomics-based research is not only the processing of large data but also modeling the complexity of features that are sources of variation across samples, which is required for an accurate statistical analysis. Therefore, our goal is to foster access for wet lab researchers to bioinformatics tools, in order to enhance their ability to explore biological aspects and validate hypotheses with robust analysis. In this context, user-friendly interfaces can enable researchers to apply computational biology methods without requiring bioinformatics expertise. Such bespoke platforms can improve the quality of the findings by allowing the researcher to freely explore the data and test a new hypothesis with independence. Simplicity DiffExpress is a data-driven software platform dedicated to enabling non-bioinformaticians to take ownership of the differential expression analysis (DEA) step in a transcriptomics experiment while presenting the results in a comprehensible layout, which supports an efficient results exploration, information storage, and reproducibility. Simplicity DiffExpress' key component is the bespoke statistical model validation that guides the user through any necessary alteration in the dataset or model, tackling the challenges behind complex data analysis. The software utilizes edgeR, and it is implemented as part of the SimplicityTM platform, providing a dynamic interface, with well-organized results that are easy to navigate and are shareable. Computational biologists and bioinformaticians can also benefit from its use since the data validation is more informative than the usual DEA resources. Wet-lab collaborators can benefit from receiving their results in an organized interface. Simplicity DiffExpress is freely available for academic use, and it is cloud-based (https://simplicity.nsilico.com/dea).

Effects of Acute Aerobic Exercise on Rats Serum Extracellular Vesicles Diameter, Concentration and Small RNAs Content.

Physical exercise stimulates organs, mainly the skeletal muscle, to release a broad range of molecules, recently dubbed exerkines. Among them, RNAs, such as miRNAs, piRNAs, and tRNAs loaded in extracellular vesicles (EVs) have the potential to play a significant role in the way muscle and other organs communicate to translate exercise into health. Low, moderate and high intensity treadmill protocols were applied to rat groups, aiming to investigate the impact of exercise on serum EVs and their associated small RNA molecules. Transmission electron microscopy, resistive pulse sensing, and western blotting were used to investigate EVs morphology, size distribution, concentration and EVs marker proteins. Small RNA libraries from EVs RNA were sequenced. Exercise did not change EVs size, while increased EVs concentration. Twelve miRNAs were found differentially expressed after exercise: rno-miR-128-3p, 103-3p, 330-5p, 148a-3p, 191a-5p, 10b-5p, 93-5p, 25-3p, 142-5p, 3068-3p, 142-3p, and 410-3p. No piRNA was found differentially expressed, and one tRNA, trna8336, was found down-regulated after exercise. The differentially expressed miRNAs were predicted to target genes involved in the MAPK pathway. A single bout of exercise impacts EVs and their small RNA load, reinforcing the need for a more detailed investigation into EVs and their load as mediators of health-promoting exercise.

Differential Expression of microRNAs in Thymic Epithelial Cells from Trypanosoma cruzi Acutely Infected Mice: Putative Role in Thymic Atrophy.

A common feature seen in acute infections is a severe atrophy of the thymus. This occurs in the murine model of acute Chagas disease. Moreover, in thymuses from Trypanosoma cruzi acutely infected mice, thymocytes exhibit an increase in the density of fibronectin and laminin integrin-type receptors, with an increase in migratory response ex vivo. Thymic epithelial cells (TEC) play a major role in the intrathymic T cell differentiation. To date, the consequences of molecular changes promoted by parasite infection upon thymus have not been elucidated. Considering the importance of microRNA for gene expression regulation, 85 microRNAs (mRNAs) were analyzed in TEC from T. cruzi acutely infected mice. The infection significantly modulated 29 miRNAs and modulation of 9 was also dependent whether TEC sorted out from the thymus exhibited cortical or medullary phenotype. In silico analysis revealed that these miRNAs may control target mRNAs known to be responsible for chemotaxis, cell adhesion, and cell death. Considering that we sorted TEC in the initial phase of thymocyte loss, it is conceivable that changes in TEC miRNA expression profile are functionally related to thymic atrophy, providing new clues to better understanding the mechanisms of the thymic involution seen in experimental Chagas disease.

Modulação da expressão gênica no timo: uma perspectiva integrativa da regulação gênica no desenvolvimento de timócitos e involução tímica decorrente de infecção / Gene expression modulation in thymus: an integrative perspective of the gene regulation during thymocyte development and thymic involution due to infection

O timo é um órgão linfoide primário no qual ocorre o desenvolvimento de células T. Distúrbios nesse processo podem levar a doenças diversas inclusive à autoimunidade. [...] Nesse sentido, essa tese aborda os aspectos moleculares envolvidos na linfopoiese T sob a perspectiva das células progenitoras e das TEC. Nosso estudod e meta-análise utilizando microarranjos de timócitos em estágios de desenvolvimento definidos pela expressão de moléculas CD4 e CD8 permitiu a identificação de 14 agrupamentos gênicos,os quais se relacionam intimamente às etapas do desenvolvimento dessas células. A modulaçãodesses genes está vinculada à sinalização via receptor de células T (TCR) e pode sercorrelacionada com a afinidade pelo complexo formado entre peptídeo e o complexo principal dehistocompatibilidade (MHC). Destacam-se ainda, vias de sinalização relacionadas a outrosreceptores de superfície, evidenciando a importância dos estímulos externos no desenvolvimentode células T. Através da integração de dados, nós identificamos as vias e moléculas com maiorimpacto para a diferenciação de timócitos, bem como complementamos a compreensão de comoos eventos envolvidos nesse processo se organizam temporalmente, apresentando umaperspectiva global das ondas de modulação gênica. Já em outra perspectiva, estudamos aregulação gênica em TEC comparando células corticais e medulares, mas também focamos nainvolução tímica, uma resposta natural a diversas fontes de estresse e ao envelhecimento.Nessa tese, esse fenômeno foi estudado em modelos experimentais de fase aguda da doençade Chagas, pois existem evidências de que as alterações no timo durante a fase aguda podemser responsáveis pelo desfecho da doença anos mais tarde...

The thymus is a primary lymphoid organ, where the T cell development takes place. Disturbanceson this process can lead to a diversity of diseases, including autoimmunity. [...] In this sense, this thesis addresses the molecular aspects involved on Tlymphopoiesis under the progenitor cells and TEC perspective. Our meta-analysis study usingmicroarrays of thymocytes sorted based on the developmental stages defined by the CD4 andCD8 expression allowed to identify 14 gene clusters, which are closely related to thedevelopmental stages of these cells. Those genes modulation is linked to the T cell receptor(TCR) signaling pathway and it can be related to the TCR affinity for the complex formed betweenthe peptide and the major histocompatibility complex (MHC). Moreover, other signaling pathwaysrelated to surface receptors stand out, highlighting the importance of external stimuli on the T celldevelopment. Through data integration, we identify pathways and molecules that have morerelevant impact on the thymocyte differentiation, as well as, we improved the comprehension onhow the events are organized temporally, providing a global perspective on the gene modulationwaves. In another perspective, we have studied the gene regulation on TEC by comparingcortical and medullary cells, but also focused on thymic involution, a natural response to varioussources of stress and aging. In this thesis, this phenomenon was studied using Chagas' diseaseacute phase experimental model because there are evidences showing that the thymic alterationsduring the acute phase can be responsible for the disease outcome years later...