Exosomal Osteoclast-Derived miRNA in Rheumatoid Arthritis: From Their Pathogenesis in Bone Erosion to New Therapeutic Approaches.

Rheumatoid arthritis (RA) is an autoimmune disease that causes inflammation, pain, and ultimately, bone erosion of the joints. The causes of this disease are multifactorial, including genetic factors, such as the presence of the human leukocyte antigen (HLA)-DRB1*04 variant, alterations in the microbiota, or immune factors including increased cytotoxic T lymphocytes (CTLs), neutrophils, or elevated M1 macrophages which, taken together, produce high levels of pro-inflammatory cytokines. In this review, we focused on the function exerted by osteoclasts on osteoblasts and other osteoclasts by means of the release of exosomal microRNAs (miRNAs). Based on a thorough revision, we classified these molecules into three categories according to their function: osteoclast inhibitors (miR-23a, miR-29b, and miR-214), osteoblast inhibitors (miR-22-3p, miR-26a, miR-27a, miR-29a, miR-125b, and miR-146a), and osteoblast enhancers (miR-20a, miR-34a, miR-96, miR-106a, miR-142, miR-199a, miR-324, and miR-486b). Finally, we analyzed potential therapeutic targets of these exosomal miRNAs, such as the use of antagomiRs, blockmiRs, agomiRs and competitive endogenous RNAs (ceRNAs), which are already being tested in murine and ex vivo models of RA. These strategies might have an important role in reestablishing the regulation of osteoclast and osteoblast differentiation making progress in the development of personalized medicine.

Variable Intrinsic Expression of Immunoregulatory Biomarkers in Breast Cancer Cell Lines, Mammospheres, and Co-Cultures.

Advances in immunotherapy have increased interest in knowing the role of the immune system in breast cancer (BC) pathogenesis. Therefore, immune checkpoints (IC) and other pathways related to immune regulation, such as JAK2 and FoXO1, have emerged as potential targets for BC treatment. However, their intrinsic gene expression in vitro has not been extensively studied in this neoplasia. Thus, we evaluated the mRNA expression of tumor-cell-intrinsic CTLA-4, PDCD1 (PD1), CD274 (PD-L1), PDCD1LG2 (PD-L2), CD276 (B7-H3), JAK2, and FoXO1 in different BC cell lines, derived mammospheres, and co-cultures with peripheral blood mononuclear cells (PBMCs) by real-time quantitative polymerase chain reaction (qRT-PCR). Our results showed that intrinsic CTLA-4, CD274 (PD-L1), and PDCD1LG2 (PD-L2) were highly expressed in triple-negative cell lines, while CD276 was predominantly overexpressed in luminal cell lines. In contrast, JAK2 and FoXO1 were under-expressed. Moreover, high levels of CTLA-4, PDCD1 (PD1), CD274 (PD-L1), PDCD1LG2 (PD-L2), and JAK2 were found after mammosphere formation. Finally, the interaction between BC cell lines and peripheral blood mononuclear cells (PBMCs) stimulates the intrinsic expression of CTLA-4, PCDC1 (PD1), CD274 (PD-L1), and PDCD1LG2 (PD-L2). In conclusion, the intrinsic expression of immunoregulatory genes seems very dynamic, depending on BC phenotype, culture conditions, and tumor-immune cell interactions.

Gamificación educativa en el laboratorio de biología celular / Educational gamification in the cell biology laboratory

La incorporación de estrategias de gamificación en la docencia se ha descrito como una herramienta para aumentar la motivación y el compromiso de los alumnos con la materia. Bajo esta premisa, se ha desarrollado una experiencia de innovación educativa mediante la plataforma Kahoot! en la primera y última práctica de laboratorio de la asignatura de Biología Celular del Grado en Biología. Los participantes fueron 135 alumnos repartidos en 12 grupos de laboratorio, que se dividieron entre experimentales y controles. Todos los grupos resolvieron un cuestionario en papel acerca de los conceptos explicados en clase, al finalizar ambas prácticas (post-test), pero sólo aquellos grupos experimentales resolvían un cuestionario antes de la clase (pre-test). Antes de la primera práctica, los alumnos de los grupos experimentales respondieron al pre-test mediante el Kahoot! Sin embargo, para la última práctica algunos grupos lo resolvieron jugando al Kahoot! y otros, con papel y bolígrafo. Los resultados mostraron que aquellos alumnos que fueron seleccionados para jugar a Kahoot!, obtuvieron un mayor número de aciertos en el test realizado tras la sesión práctica (post-test) con respecto a aquellos que no resolvieron ningún pre-test o, que lo hicieron de un modo clásico. Por lo tanto, nuestros resultados sugieren que implementar la jugabilidad en la docencia incrementa considerablemente la motivación del alumnado debido, probablemente, a cambios fisiológicos experimentados por el cerebro durante el juego y a la creación de un clima positivo, que facilitan el proceso de aprendizaje.

SUMMARY: The incorporation of gamification strategies in teaching has been described as a tool to increase the motivation and engagement of students with the subject. Under this premise, an educational innovation experience has been developed using the Kahoot! platform in the first and last laboratory practice of the Cell Biology course of the Biology degree. The participants were 135 students divided into 12 laboratory groups, which were divided into experimental and control groups. All groups solved a questionnaire on paper about the concepts explained in class, at the end of both practices (post-test), but only the experimental groups solved a questionnaire before the class (pre-test). Before the first practice, students in the experimental groups answered the pre-test using Kahoot! However, for the last practice, some groups solved it by playing Kahoot! and others with pen and paper. The results showed that those students who were selected to play Kahoot! obtained a higher number of correct answers in the test performed after the practical session (post-test) than those who did not solve any pre- test or who did it in a classical way. Therefore, our results suggest that implementing gamification in teaching considerably increases student motivation, probably due to physiological changes experienced by the brain during the game and the creation of a positive climate, which facilitates the learning process.

Antioxidant Supplementation Modulates Neutrophil Inflammatory Response to Exercise-Induced Stress.

The aim of the present report was to evaluate the inflammatory response to a 2000-m running test considering neutrophil myeloperoxidase as an inflammatory marker, and to verify if supplements rich in antioxidants could modulate Post-test antioxidant and anti-inflammatory responses. To this end, a 21-day homogenization period was carried out with three groups: a control group, a supplemented group taking an almond beverage enriched with vitamins C and E and a third group consuming the same beverage but enriched with Lippia citriodora extract. At the end of this period, participants performed a 2000-m run, and blood samples were obtained the day before and immediately after the running test. Plasma and neutrophils were isolated. As a result, plasma creatine kinase and myoglobin increased, indicating Post-test muscle damage. Plasma oxidative markers were increased in all groups, except in the group supplemented with the almond beverage. Neutrophil antioxidant enzymes were significantly increased only in the control group, suggesting an antioxidant effect of the supplements provided in the other groups. Myeloperoxidase activity was significantly increased after the test in the control group, while increased enzyme levels were detected in plasma of the supplement groups. Therefore, antioxidant consumption seems to favour myeloperoxidase release. The connection of this observation with post-exercise recovery will require further investigation.

Differences of Clonogenic Mesenchymal Stem Cells on Immunomodulation of Lymphocyte Subsets.

Mesenchymal stem cells (MSC) are a widely used population in cell therapy for their ability to differentiate into distinct tissues and more lately, for their immunomodulatory properties. However, the use of heterogeneous populations could be responsible for the nondesired outcomes reflected in the literature. Here, we analyse the different capacities of five one-cell-derived MSC clones to exert their immunomodulation ex vivo. We assessed proliferation assays in cocultures of MSC clones and purified cluster of differentiation (CD)3+, CD4+, or CD8+ lymphocytes; analysed the regulatory T (Treg) cells fold change rate; determined the effects on viability of peripheral blood mononuclear cells (PBMC); and also measured the coculture cytokine profiles (Th1/Th2). Conditioned media (CM) of different clones were also used to perform both proliferation assays and to analyse Treg fold change. The five clones analysed in this work were able to generate heterogeneous environments. Different clones inhibited proliferation of CD3+ and CD4+ lymphocytes, with different intensities. Surprisingly, all clones promoted proliferation of CD8+ lymphocytes. Different MSC clones and their CM were able to increase the number of Treg with different intensities. Finally, different clones also promoted different effects on the viability of PBMC treated with ultraviolet light. Considering all these data together, it seems that different clones, even from the same donor, can promote a wide spectrum of responses from anti-inflammatory to proinflammatory character. This fact may be important to standardise the design of personalized cell therapy protocols, thus diminishing the aforementioned undesired outcomes existing nowadays in this type of therapies.

Effect of a 2000-m running test on antioxidant and cytokine response in plasma and circulating cells.

Exercise intensity usually correlates with increased oxidative stress and enhanced cytokine production. However, it is unknown if all types of exercise that induce muscle damage can cause a parallel response in the oxidation balance and cytokine production. To this end, the effect of a 2000-m running test in a group of volunteers that regularly train in aerobic routines was studied. Different circulating parameters were measured, oxidative stress markers (protein carbonyls and malondialdehyde), antioxidant enzyme activity, and cytokine levels in plasma as well as in the main circulating cells of blood samples obtained in basal conditions and after test execution. As a result, the test caused muscle damage evidenced by an increase in circulating creatine kinase and myoglobin. This was accompanied by an increase in protein carbonyls in plasma and peripheral blood mononuclear cells. Activities of antioxidant enzymes (catalase, glutathione peroxidase and reductase, superoxide dismutase) were elevated in peripheral blood mononuclear cells, neutrophils, and erythrocytes after the test. Regarding cytokine production, interleukin-6, interleukin-8, interleukin-10, and tumor necrosis factor-α exhibited no significant changes after the test. Results suggest that this short but intense running exercise (2000 m) can induce muscle damage and elicit a good balance between oxidant/antioxidant responses with no changes in the circulating concentration of pro-inflammatory cytokines.

CD44 induces FOXP3 expression and is related with favorable outcome in breast carcinoma.

We studied the relationship between CD44 and Forkhead box P3 (FOXP3) gene expression in cell lines and breast carcinomas and their association with clinicopathological variables and patient outcome. We assessed messenger RNA (mRNA) expression of CD44 and FOXP3 by quantitative real-time PCR and determined the number of FOXP3+ Tregs by immunohistochemistry in 264 breast cancer specimens. CD44 was stimulated with hyaluronan treatment, and the accompanying changes in FOXP3 mRNA expression in breast cancer cell lines representing breast cancer subtype were assessed. We found that lower CD44 expression correlated with the presence of necrosis, lymph-vascular invasion, grade 3 tumors, and aggressive phenotype (HER2 and basal-like). FOXP3 mRNA correlated positively with CD44 mRNA expression and Treg content. Moreover, stimulation of CD44 expression by hyaluronan in cell lines increased FOXP3 expression, which supports that their regulation is associated. Survival analysis revealed that low CD44 expression is associated with higher frequency of recurrence. Our findings indicate that CD44 has a regulatory role in FOXP3 expression and is associated with good prognostic factors in breast cancer.

Carbon and nitrogen limitation increase chitosan antifungal activity in Neurospora crassa and fungal human pathogens.

Chitosan permeabilizes plasma membrane and kills sensitive filamentous fungi and yeast. Membrane fluidity and cell energy determine chitosan sensitivity in fungi. A five-fold reduction of both glucose (main carbon (C) source) and nitrogen (N) increased 2-fold Neurospora crassa sensitivity to chitosan. We linked this increase with production of intracellular reactive oxygen species (ROS) and plasma membrane permeabilization. Releasing N. crassa from nutrient limitation reduced chitosan antifungal activity in spite of high ROS intracellular levels. With lactate instead of glucose, C and N limitation increased N. crassa sensitivity to chitosan further (4-fold) than what glucose did. Nutrient limitation also increased sensitivity of filamentous fungi and yeast human pathogens to chitosan. For Fusarium proliferatum, lowering 100-fold C and N content in the growth medium, increased 16-fold chitosan sensitivity. Similar results were found for Candida spp. (including fluconazole resistant strains) and Cryptococcus spp. Severe C and N limitation increased chitosan antifungal activity for all pathogens tested. Chitosan at 100 µg ml(-1) was lethal for most fungal human pathogens tested but non-toxic to HEK293 and COS7 mammalian cell lines. Besides, chitosan increased 90% survival of Galleria mellonella larvae infected with C. albicans. These results are of paramount for developing chitosan as antifungal.