Janus kinase inhibitors modify the fatty acid profile of extracellular vesicles and modulate the immune response.

Background: Janus kinase inhibitors (jakinibs) are immunomodulators used for treating malignancies, autoimmune diseases, and immunodeficiencies. However, they induce adverse effects such as thrombosis, lymphocytosis, and neutropenia that could be mediated by extracellular vesicles (EVs). These particles are cell membrane-derived structures that transport cellular and environmental molecules and participate in intercellular communication. Jakinibs can modify the content of EVs and enable them to modulate the activity of different components of the immune response. Objective: to evaluate the interactions between immune system components of healthy individuals and EVs derived from monocytic and lymphoid lineage cells generated in the presence of baricitinib (BARI) and itacitinib (ITA) and their possible effects. Methods: EVs were isolated from monocytes (M) and lymphocytes (L) of healthy individuals, as well as from U937 (U) and Jurkat (J) cells exposed to non-cytotoxic concentrations of BARI, ITA, and dimethyl sulfoxide (DMSO; vehicle control). The binding to and engulfment of EVs by peripheral blood leukocytes of healthy individuals were analyzed by flow cytometry using CFSE-stained EVs and anti-CD45-PeCy7 mAb-labeled whole blood. The effect of EVs on respiratory burst, T-cell activation and proliferation, cytokine synthesis, and platelet aggregation was evaluated. Respiratory burst was assessed in PMA-stimulated neutrophils by the dihydrorhodamine (DHR) test and flow cytometry. T-cell activation and proliferation and cytokine production were assessed in CFSE-stained PBMC cultures stimulated with PHA; expression of the T-cell activation markers CD25 and CD69 and T-cell proliferation were analyzed by flow cytometry, and the cytokine levels were quantified in culture supernatants by Luminex assays. Platelet aggregation was analyzed in platelet-rich plasma (PRP) samples by light transmission aggregometry. The EVs' fatty acid (FA) profile was analyzed using methyl ester derivatization followed by gas chromatography. Results: ITA exposure during the generation of EVs modified the size of the EVs released; however, treatment with DMSO and BARI did not alter the size of EVs generated from U937 and Jurkat cells. Circulating neutrophils, lymphocytes, and monocytes showed a 2-fold greater tendency to internalize ITA-U-EVs than their respective DMSO control. The neutrophil respiratory burst was attenuated in greater extent by M-EVs than by L-EVs. Autologous ITA-M-EVs reduced T-cell proliferation by decreasing IL-2 levels and CD25 expression independently of CD69. A higher accumulation of pro-inflammatory cytokines was observed in PHA-stimulated PBMC cultures exposed to M-EVs than to L-EVs; this difference may be related to the higher myristate content of M-EVs. Platelet aggregation increased in the presence of ITA-L/M-EVs by a mechanism presumably dependent on the high arachidonic acid content of the vesicles. Conclusions: Cellular origin and jakinib exposure modify the FA profile of EVs, enabling them, in turn, to modulate neutrophil respiratory burst, T-cell proliferation, and platelet aggregation. The increased T-cell proliferation induced by BARI-L/M-EVs could explain the lymphocytosis observed in patients treated with BARI. The higher proportion of arachidonic acid in the FA content of ITA-L/M-EVs could be related to the thrombosis described in patients treated with ITA. EVs also induced a decrease in the respiratory burst of neutrophils.

Circulating extracellular vesicles in Systemic Lupus Erythematosus: physicochemical properties and phenotype.

OBJECTIVE: This study aimed to identify the physicochemical and phenotypic characteristics of circulating Extracellular Vesicles (EVs) in the plasma of patients with SLE, with or without Lupus Nephritis (LN), and their potential utility as disease biomarkers. METHODS: Plasma-circulating EVs were concentrated using differential centrifugation from adult female patients (n=38) who met the 'American College of Rheumatology/European Alliance of Associations for Rheumatology 2019' criteria for SLE diagnosis with (LN) or without LN (nLN), confirmed by renal biopsy. Controls (n=18) were healthy volunteers matched by gender and similar age. The structure, size and Energy Dispersion Spectrum (EDS) of EVs were observed by electron microscopy. The surface charge and size distribution were evaluated using dynamic light scattering. The counts and phenotype of EVs from patients (SLE-EVs) and controls (Ctrl-EVs) were obtained using flow cytometry. Non-parametric statistical tests and exploratory analysis of multiple variables were performed. The discriminatory power of some variables as potential biomarkers of the disease was also evaluated. RESULTS: Circulating EVs were heterogeneous in morphology and size, but SLE-EVs reached larger diameters than Ctrl-EVs (p<0.0001). Small SLE-EVs and large SLE-EVs were increased compared with Ctrl-EV (p<0.0001 and p<0.05, respectively). Likewise, patients with SLE (LN or nLN) had higher concentrations of large EVs compared with controls (p<0.001 and p<0.0001, respectively). SLE-EVs showed a different EDS (p<0.001) and were less electronegative (p<0.0001) than Ctrl-EVs. EV-CD45+, EV-CD14+ and EV-IgM+ were more frequent in patients with SLE compared with controls (p<0.001, p<0.05 and p<0.001, respectively). The concentrations of large EVs and EV-IgM+ allowed better discrimination of patients from controls. CONCLUSIONS: Plasma-circulating EVs from patients with SLE with and without nephritis are increased in peripheral blood and have different physicochemical properties than controls. Characteristics of EVs such as larger size and the presence of IgM on the surface could help discriminate patients from controls.

Lippia origanoides Essential Oil or Thymol in Combination with Fluconazole Produces Damage to Cells and Reverses the Azole-Resistant Phenotype of a Candida tropicalis Strain.

Candida tropicalis is one of the most pathogenic species within the genus. Increased antifungal resistance has been reported, which is in part due to the organism's ability to form biofilms. In natural products derived from plants, such as essential oils (EOs) or their major components, there is significant potential to develop new antifungals or to both enhance the efficacy and reduce the toxicity of conventional antifungals. This study aimed to evaluate the effect of combining an EO of Lippia origanoides or thymol with fluconazole on an azole-resistant C. tropicalis strain. Synergism was observed in the combination of fluconazole with the EO and with thymol, and minimal inhibitory concentrations for fluconazole decreased at least 32-fold. As a consequence of the synergistic interactions, mitochondrial membrane potential was reduced, and mitochondrial superoxide production increased. Alteration in nuclear morphology, cell surface, and ultrastructure was also observed. In conclusion, the synergistic interaction between L. origanoides EO or thymol with fluconazole reverted the azole-resistant C. tropicalis phenotype. These findings suggest that L. origanoides EO or thymol alone, or in combination with fluconazole, have the potential for development as antifungal therapies for this yeast, including resistant strains.

Role of aspirin-triggered lipoxin A4, aspirin, and salicylic acid in the modulation of the oxidative and inflammatory responses induced by plasma from women with pre-eclampsia.

PROBLEM: Oxidative stress and inflammation are key events leading to pre-eclampsia, involved in several maternal deaths. Low doses of acetylsalicylic acid (ASA) are used in the prevention and treatment of pre-eclampsia. The synthesis of aspirin-triggered lipoxin (ATL) by cyclooxygenase-2 acetylation is an alternative mechanism of ASA, which could explain the effectiveness of ASA treatments. The aim of this study was to evaluate the role of ASA, salicylates, and ATL in the modulation of the oxidative and inflammatory responses induced by plasma from women with pre-eclampsia. METHOD OF STUDY: Plasma from 14 women with pre-eclampsia and 17 normotensive pregnant women was probed for inducing oxidative and inflammatory responses on endothelial cells and U937 promonocytes. The role of ATL, ASA, and salicylic acid (SA) on these events was evaluated. RESULTS: Plasma from women with pre-eclampsia induced TBARS and nitrotyrosine production on endothelial and U937 cells. Pre-treatment with both ATL and ASA decreased the TBARS production, while ATL decreased the nitrotyrosine. Pre-eclamptic plasma augmented the translocation of NF-kB on U937 cells, which decreased by a high dose of ASA and SA. Finally, the pre-eclamptic plasma increased the adhesion of leukocytes-PMN and monocytes-to endothelium, and we were able to determine a state of resolution of inflammation, since ATL decreased the PMN adhesion, and conversely, it increased the monocytes adhesion to endothelium. CONCLUSION: Together, these results suggest that ATL could explain the beneficial actions of ASA and support further research on mechanisms, real efficacy, and rational use of ASA in pre-eclampsia.

Polyacrylic acid-coated iron oxide nanoparticles could be a useful tool for tracking inflammatory monocytes.

AIM: To establish the effect of poly(acrylic acid)-coated iron oxide nanoparticles (PAC-IONs) and later exposure to a magnetic field on the differentiation of mononuclear phagocytes into macrophages. METHODS: By flow cytometry, cell death was evaluated with DIOC6 and PI, Poly (ADP-ribose) Polymerases (PARP) fragmentation, H2AX phosphorylation and TUNEL assay. Cytokines by Cytokine bead array and the intracellular amount of iron by atomic absorption spectrometry. RESULTS: PAC-IONs did not induce apoptosis, modify the cell membrane integrity or alter the mitochondrial membrane potential. They did not affect the cell morphology, the pattern of cytokine accumulation or the activating role of differentiation of mononuclear phagocytes into macrophages on the proliferation of autologous T cells. CONCLUSION: This evidence indicates that the PAC-IONs are safe and biocompatible. Moreover, the selectivity of the PAC-IONs for mononuclear phagocytes, as well as their increased uptake by non-classical monocytes, warrant future research with a view to their use as a contrast agent, a useful tool for in vivo tracking of tissue-infiltrating mononuclear phagocytes.

Role of aspirin-triggered lipoxin A4, aspirin, and salicylic acid in the modulation of the oxidative and inflammatory responses induced by plasma from women with pre-eclampsia

Problem Oxidative stress and inflammation are key events leading to pre-eclampsia, involved in several maternal deaths. Low doses of acetylsalicylic acid (ASA) are used in the prevention and treatment of pre-eclampsia. The synthesis of aspirin-triggered lipoxin (ATL) by cyclooxygenase-2 acetylation is an alternative mechanism of ASA, which could explain the effectiveness of ASA treatments. The aim of this study was to evaluate the role of ASA, salicylates, and ATL in the modulation of the oxidative and inflammatory responses induced by plasma from women with pre-eclampsia. Method of study Plasma from 14 women with pre-eclampsia and 17 normotensive pregnant women was probed for inducing oxidative and inflammatory responses on endothelial cells and U937 promonocytes. The role of ATL, ASA, and salicylic acid (SA) on these events was evaluated. Results Plasma from women with pre-eclampsia induced TBARS and nitrotyrosine production on endothelial and U937 cells. Pre-treatment with both ATL and ASA decreased the TBARS production, while ATL decreased the nitrotyrosine. Pre-eclamptic plasma augmented the translocation of NF-kB on U937 cells, which decreased by a high dose of ASA and SA. Finally, the pre-eclamptic plasma increased the adhesion of leukocytes—PMN and monocytes—to endothelium, and we were able to determine a state of resolution of inflammation, since ATL decreased the PMN adhesion, and conversely, it increased the monocytes adhesion to endothelium. Conclusion Together, these results suggest that ATL could explain the beneficial actions of ASA and support further research on mechanisms, real efficacy, and rational use of ASA in pre-eclampsia.

Role of aspirin-triggered lipoxin A4, aspirin, and salicylic acid in the modulation of the oxidative and inflammatory responses induced by plasma from women with pre-eclampsia

Problem Oxidative stress and inflammation are key events leading to pre-eclampsia, involved in several maternal deaths. Low doses of acetylsalicylic acid (ASA) are used in the prevention and treatment of pre-eclampsia. The synthesis of aspirin-triggered lipoxin (ATL) by cyclooxygenase-2 acetylation is an alternative mechanism of ASA, which could explain the effectiveness of ASA treatments. The aim of this study was to evaluate the role of ASA, salicylates, and ATL in the modulation of the oxidative and inflammatory responses induced by plasma from women with pre-eclampsia. Method of study Plasma from 14 women with pre-eclampsia and 17 normotensive pregnant women was probed for inducing oxidative and inflammatory responses on endothelial cells and U937 promonocytes. The role of ATL, ASA, and salicylic acid (SA) on these events was evaluated. Results Plasma from women with pre-eclampsia induced TBARS and nitrotyrosine production on endothelial and U937 cells. Pre-treatment with both ATL and ASA decreased the TBARS production, while ATL decreased the nitrotyrosine. Pre-eclamptic plasma augmented the translocation of NF-kB on U937 cells, which decreased by a high dose of ASA and SA. Finally, the pre-eclamptic plasma increased the adhesion of leukocytes—PMN and monocytes—to endothelium, and we were able to determine a state of resolution of inflammation, since ATL decreased the PMN adhesion, and conversely, it increased the monocytes adhesion to endothelium. Conclusion Together, these results suggest that ATL could explain the beneficial actions of ASA and support further research on mechanisms, real efficacy, and rational use of ASA in pre-eclampsia.

Entendiendo los monocitos para rastrear la tuberculosis / Understanding monocytes to trace tuberculosis

Debido al gran número de casos nuevos de tuberculosis, la emergencia de cepas resistente y las comorbilidades que comprometen la función del sistema inmune [1], esta condición patológica tiene cada vez mayor impacto en el ámbito global. A pesar de las múltiples investigaciones, la complejidad del problema y los impactos sociales y económicos hacen de la tuberculosis una enfermedad que se considera devastadora. Según la Organización Mundial de la Salud (OMS) se estima que una cuarta parte de la población mundial tiene tuberculosis latente; término aplicado para las personas infectadas por Mycobacterium tuberculosis que aún no han enfermado ni pueden transmitir la infección. La tuberculosis, durante muchos años, y también según las estadísticas de la OMS, está dentro de las diez primeras causas de muerte en el mundo [2]. Si se contrasta con otras enfermedades infecciosas, también devastadoras, como la diarrea y las enfermedades respiratorias, que pueden ser originadas por múltiples agentes etiológicos, la tuberculosis es la enfermedad más importante causada por un único agente etiológico. La infección usualmente comienza por el tracto respiratorio, donde las bacterias inhaladas pueden ser fagocitadas por los macrófagos residentes del alvéolo, los cuales son los responsables de activar los mecanismos tempranos de la respuesta innata [3]. Esta respuesta compromete otros tipos de células como las dendríticas y los monocitos, reclutados de la corriente sanguínea, e incluso subpoblaciones de NK [4,5] y neutrófilos [6,7]. Las interacciones iniciales entre la micobacteria y las células hospederas fagocíticas han sido consideradas como eventos capitales en las fases ulteriores de la respuesta inmune [8,9]. Por ello, dadas las restricciones para acceder de manera expedita a muestras de pulmón, estas interacciones han sido estudiadas, por ejemplo, en nuestro caso, utilizando fagocitos obtenidos principalmente de sangre periférica, promonocitos humanos [10-13] y macrófagos esplénicos tanto humanos[14,15] como múridos [16]. Nuestras observaciones han hecho evidentes varias diferencias entre los fagocitos mononucleares de los pacientes con tuberculosis y los individuos sanos. Una de las más notorias consiste en que los monocitos de los pacientes, cuando son infectados in vitro con Mycobacterium tuberculosis H37Rv o tratados con derivado proteico purificado (PPD), una fracción de ellos muere con evidentes alteraciones en la membrana celular y otra con daño mitocondrial, exposición de la fosfatidilserina, activación de caspasas y daño en el ADN. En el caso de los monocitos de los controles sanos se observa fundamentalmente muerte con daño mitocondrial y una escasa proporción de células con daño en la membrana [11,12]

Estrategias alternativas para el diagnóstico de tuberculosis: una opción para los pacientes paucibacilares / alternative strategies for the tuberculosis diagnosis: an option for paucibacillary patients

el diagnóstico de la tuberculosis ha estado basado en la detección directa de la micobacteria; sin embargo, se estima que este se puede lograr solamente en el 10% de los casos y requiere que se combine con métodos confirmatorios como el cultivo, el cual puede tomar varias semanas para que el crecimiento sea evidente. Los métodos basados en la amplificación de la secuencia ácidos nucleicos muestran sensibilidad y especificidad altas, pero no siempre son accesibles a todos los laboratorios debido a sus requerimientos de infraestructura y el costo de los insumos. Las limitaciones para el diagnóstico hacen que se busque continuamente metabolitos micobacterianos, mediante diferentes aproximaciones, que sean, ulteriormente, fáciles de rastrear en condiciones muy básicas de laboratorio. En esta revisión se incluyen algunas de las aproximaciones metodológicas basadas en la detección de derivados micobacterianos y su valor como herramienta para el rastreo de la micobacteria

The diagnosis of tuberculosis has been based on the direct detection of mycobacteria. However, it is estimated that only can be achieved in 10% of the cases and it is necessary to combine with confirmatory methods such as culture that may take several weeks to growth been evident. Methods based on sequence amplification of nucleic acids show high sensitivity and specificity, but are not always accessible to all laboratories due to infrastructure requirements and the cost of inputs. The limitations for the diagnosis induce to a continued research for mycobacterial metabolites by different approaches that are later easy to trace in very basic laboratory conditions. This review includes some of the methodological approaches based on mycobacterial derivatives and their value as a tool to detect the mycobacteria

Subpoblaciones de linfocitos B y su expresión de CD1d en pacientes con lupus eritematoso sistémico / B cell subsets and their expression of CD1d in patients with systemic lupus erythematosus

Resumen Introducción: Los linfocitos B (LB) se consideran el centro de la desregulación inmune en pacientes con lupus eritematoso sistémico (LES), principalmente, por su producción de autoanticuerpos. Recientemente, se demostró la existencia de LB, incluidos en los B transicionales, con capacidad reguladora (Breg) y fenotipo CD19+CD24hiCD38hi. En humanos se demostró la importancia de CD80 y CD86 en su función reguladora. El papel de CD1d aún no ha sido evaluado. Objetivo: Evaluar la frecuencia de LB maduros, memoria y transicionales, en controles y pacientes con LES, además de la expresión de CD1d y correlacionarla con la actividad de la enfermedad medida por SLEDAI (Systemic Lupus Erythematosus Disease Activity Index). Materiales y métodos: Se evaluó por citometría de flujo la frecuencia de subpoblaciones de LB basados en la expresión de CD19, CD24 y CD38, además de CD1d, en controles con otras enfermedades autoinmunes (OEA), individuos sanos y pacientes con LES, y se correlacionó con SLEDAI. Resultados: Se evidenció una disminución significativa en el porcentaje de LB de memoria en pacientes LES y OEA, sin alteraciones en las subpoblaciones de LB maduros y transicionales. La expresión de CD1d no evidenció diferencias significativas en ninguna de las subpoblaciones ni se correlacionó con SLEDAI. Conclusión: La disminución de la subpoblación de memoria fue previamente descrita en LES y se ha asociado a algunos tipos de tratamiento. Aunque CD1d se ha asociado a la función de Breg en murinos, no hubo diferencias significativas en su expresión en las subpoblaciones y queda por clarificar su papel en la función de las Breg humanas.

Abstract Introduction: B lymphocytes are considered the center of immune dysregulation in Systemic Lupus Erythematosus (SLE). It has recently been demonstrated that there is a B cell with regulatory capacities (Breg) included in transitional B lymphocytes with the phenotype CD19+CD24hiCD38hi. The importance of CD80 and CD86 in the regulatory function of the Bregs has been demonstrated in humans, but the role of CD1d has not been evaluated. Objective: To evaluate the frequency of mature, memory and transitional B cells in SLE patients and controls, the expression of CD1d among these cells, and its correlation with the activity of the disease measured using the Systemic Lupus Erythematosus Disease Activity Index (SLEDAI). Materials and methods: The frequency of the B cell subsets was evaluated by flow cytometry based on the expression of CD19, CD24 and CD38, as well as CD1d in these cells in SLE patients and controls, and were correlated with the activity of the disease measured using the SLEDAI. Results: A significant reduction in the percentage of memory B cells was observed in SLE patients and other autoimmune conditions, with no changes in the mature or transitional B cell subsets. Similarly, no significant differences were observed in the expression of CD1d in any of the subsets, nor was there any correlation with the SLEDAI. Conclusion: The reduction of the memory subset has been previously described in SLE, and has been associated with some types of treatment. The expression of CD1d in all the subsets was observed, but its role in the regulatory function of the CD19+CD24hiCD38hi cells is still not clear.

La necrosis, un mecanismo regulado de muerte celular / Necrosis, a regulated mechanism of cell death

Con base en criterios morfológicos y bioquímicos se han definido tres clases de muerte celular: apoptosis, autofagia y necrosis. La primera es una muerte celular regulada, mediada principalmente por caspasas; en la autofagia ocurre formación de vesículas que se fusionan con vacuolas hidrolíticas para degradar organelas intracelulares alteradas. En cuanto a la necrosis, se la ha definido tradicionalmente por la ruptura de la membrana citoplasmática con salida del material intracelular lo que desencadena una reacción inflamatoria localizada; los mediadores pueden variar dependiendo del tejido: lipasas, proteasas y endonucleasas. Las actividades celulares intrínsecas y los eventos que preceden al colapso celular definen el tipo de daño. Pero el hecho de que los tres tipos de muerte celular puedan coexistir y la ocurrencia de necrosis incluso en presencia de ATP hacen pensar que esta es un evento menos pasivo y que hasta cierto punto se puede regular la inducción del daño. Las alteraciones en la estructura de proteínas y en la actividad de proteasas, lipasas y endonucleasas en presencia de sus cofactores, asociadas con los mecanismos intrínsecos de regulación celular permiten pensar que cada célula puede tener su propio arsenal para producir los eventos designados como necrosis (recientemente denominada parapoptosis). En este artículo se revisan algunas de las evidencias sobre la regulación durante la necrosis en diferentes modelos celulares.

Three types of cellular death have been defined by morphological and biochemical criteria: apoptosis, necrosis and autophagy. Apoptosis is a regulated cell death, mainly mediated by caspases; autophagy induces degradation of intracellular damaged organelles through the formation of vesicles that fuse with hydrolytic vacuoles. Necrosis has been traditionally defined by the rupture the cytoplasmic membrane with subsequent release of intracellular material, triggering localized inflammatory Intrinsic cellular activities and the events preceding cellular collapse are critical to determine the type of tissue damage. The fact that all three types of cellular death can coexist in any organ and tissue with different availabilities of ATP, suggests that necrosis can be conceived as an active event and that to some extent it may be regulated. Alterations in the structure of proteins and in the activity of different proteases, lipases and nucleases, indicate that each cell may have its own arsenal to trigger the events leading to necrosis. In this article we review some of the evidences on cellular regulation during necrosis.