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Mariano García-Martínez,1,2 Luis Vázquez-Flores,1,2 Violeta D Álvarez-Jiménez,1,3 Jessica Castañeda-Casimiro,1 Miguel Ibáñez-Hernández,4 Luvia Enid Sánchez-Torres,1 Jorge Barrios-Payán,5 Dulce Mata-Espinosa,5 Sergio Estrada-Parra,1 Rommel Chacón-Salinas,1 Jeanet Serafín-López,1 Isabel Wong-Baeza,1 Rogelio Hernández-Pando,5 Iris Estrada-García11Departamento de Inmunología, Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional (IPN), Mexico City, Mexico; 2Posgrado en Inmunología, Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional (IPN), Mexico City, Mexico; 3Laboratorio de Biología Molecular y Bioseguridad Nivel 3, Centro Médico Naval (CEMENAV), Secretaría de Marina Armada de México (Semar), Mexico City, Mexico; 4Departamento de Bioquímica, Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional (IPN), Mexico City, Mexico; 5Sección de Patología Experimental, Departamento de Patología, Instituto Nacional de Ciencias Médicas y Nutrición Salvador Zubirán, Mexico City, MexicoCorrespondence: Iris Estrada-GarcíaDepartamento de Inmunología, Escuela Nacional de Ciencias Biológicas (ENCB), Instituto Politécnico Nacional (IPN), Prolongación de Carpio y Plan de Ayala s/n, Col. Santo Tomás, Ciudad de México 11340, MéxicoTel +52 555 729 6000 x 62507Fax +52 555 729 6000 x 62369Email iestrada5@hotmail.comBackground: Tuberculosis is the leading cause of death by an infectious microorganism worldwide. Conventional treatment lasts at least six months and has adverse effects; therefore, it is important to find therapeutic alternatives that reduce the bacterial load and may reduce the treatment duration. The immune response against tuberculosis can be modulated by several mechanisms, including extracellular vesicles (EVs), which are nano-sized membrane-bound structures that constitute an efficient communication mechanism among immune cells.Methods: The EVs released by the J774A.1 mouse macrophage cell line, both spontaneously (S-EV) and after infection with Mycobacterium tuberculosis H37Rv (Mtb-EV), were purified by ultra-centrifugation and size-exclusion chromatography. The size distribution and chemical composition of these EVs were evaluated, and their effect on the bacterial load and the production of cytokines was determined in both in vitro and in vivo models of M. tuberculosis infection.Results: Mtb-EV are larger than S-EV, they contain M. tuberculosis-specific antigens (not detected in EVs released from M. fortuitum-infected J774A.1 cells) and are rich in phosphatidylserine, present in their outer membrane layer. S-EV, but not Mtb-EV, reduced the bacterial load and the production of MCP-1 and TNF-α in M. tuberculosis-infected macrophages, and these effects were reversed when phosphatidylserine was blocked with annexin V. Both S-EV and Mtb-EV significantly reduced the lung bacterial load in mice infected with M. tuberculosis after 60 days of treatment, but they had no effect on survival or on the lung pneumonic area of these mice.Conclusion: J774A.1 macrophages infected with M. tuberculosis H37Rv released EVs that differed in size and phosphatidylserine content from spontaneously released EVs, and these EVs also had different biological effects: S-EV reduced the mycobacterial load and the cytokine production in vitro (through a phosphatidylserine-dependent mechanism), while both EVs reduced the lung bacterial load in vivo. These results are the basis for further experiments to evaluate whether EVs improve the efficiency of the conventional treatment for tuberculosis.Keywords: tuberculosis, extracellular vesicles, phosphatidylserine, macrophages, lung disease

Background: Cell migration is essential for the immune system and is frequently analyzed in adult non-pregnant animals but poorly explored in pregnant animals. However, a physiologic increased size in the spleen and periaortic lymph nodes had been reported in pregnant mice., Methods: Using a mouse model, we transferred PKH26-stained thymocytes and splenocytes from pregnant or non-pregnant animals to receptor mice in the presence or absence of pregnancy. Percentage of PKH-26 cells and Mean Fluorescence Intensity were calculated. Non-parametric ANOVA analysis was performed., Results: We detected that the percentage of PKH26+ thymocytes in the spleen, lymph nodes, and peripheral blood is higher in females than in males (p = 0.039). Our results showed a similar frequency of thymocytes and splenocytes from pregnant and non-pregnant mice located in receptor lymphoid organs (p > 0.05). Also, the location of marked cells was similar during the perinatal period (p > 0.05)., Conclusions: The mobility of thymocytes and splenocytes in pregnant and non-pregnant mice is similar. Therefore, we suggest that the larger size of the spleen and periaortic lymph nodes noted previously in pregnant mice could result from the retention of leukocytes in the secondary lymphoid organs., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (© 2024 The Authors. Published by Elsevier B.V.)

Mesenchymal stromal cells (MSCs) together with malignant cells present in the tumor microenvironment (TME), participate in the suppression of the antitumor immune response through the production of immunosuppressive factors, such as transforming growth factor beta 1 (TGF-β1). In previous studies, we reported that adenosine (Ado), generated by the adenosinergic activity of cervical cancer (CeCa) cells, induces the production of TGF-β1 by interacting with A 2A R/A 2B R. In the present study, we provide evidence that Ado induces the production of TGF-β1 in MSCs derived from CeCa tumors (CeCa-MSCs) by interacting with both receptors and that TGF-β1 acts in an autocrine manner to induce the expression of programmed death ligand 1 (PD-L1) in CeCa-MSCs, resulting in an increase in their immunosuppressive capacity on activated CD8+ T lymphocytes. The addition of the antagonists ZM241385 and MRS1754, specific for A 2A R and A 2B R, respectively, or SB-505124, a selective TGF-β1 receptor inhibitor, in CeCa-MSC cultures significantly inhibited the expression of PD-L1. Compared with CeCa-MSCs, MSCs derived from normal cervical tissue (NCx-MSCs), used as a control and induced with Ado to express PD-L1, showed a lower response to TGF-β1 to increase PD-L1 expression. Those results strongly suggest the presence of a feedback mechanism among the adenosinergic pathway, the production of TGF-β1, and the induction of PD-L1 in CeCa-MSCs to suppress the antitumor response of CD8+ T lymphocytes. The findings of this study suggest that this pathway may have clinical importance as a therapeutic target., (© 2024 The Authors. Cell Biochemistry and Function published by John Wiley & Sons Ltd.)

Twenty 2-(4-alkyloxyphenyl)-imidazolines and 2-(4-alkyloxyphenyl)-imidazoles were synthesized, with the former being synthesized in two steps by using MW and ultrasonication energy, resulting in good to excellent yields. Imidazoles were obtained in moderate yields by oxidizing imidazolines with MnO 2 and MW energy. In response to the urgent need to treat neglected tropical diseases, a set of 2-(4-alkyloxyphenyl)- imidazolines and imidazoles was tested in vitro on Leishmania mexicana and Trypanosoma cruzi . The leishmanicidal activity of ten compounds was evaluated, showing an IC 50 < 10 µg/mL. Among these compounds, 27 - 31 were the most active, with IC 50 values < 1 µg/mL (similar to the reference drugs). In the evaluation on epimastigotes of T. cruzi , only 30 and 36 reached an IC 50 < 1 µg/mL, showing better inhibition than both reference drugs. However, compounds 29 , 33 , and 35 also demonstrated attractive trypanocidal activities, with IC 50 values < 10 µg/mL, similar to the values for benznidazole and nifurtimox.

The thymus is the anatomical site where T cells undergo a complex process of differentiation, proliferation, selection, and elimination of autorreactive cells which involves molecular signals in different intrathymic environment. However, the immunological functions of the thymus can be compromised upon exposure to different infections, affecting thymocyte populations. In this work, we investigated the impact of malaria parasites on the thymus by using C57BL/6 mice infected with Plasmodium berghei ANKA and Plasmodium yoelii 17XL; these lethal infection models represent the most severe complications, cerebral malaria, and anemia respectively. Data showed a reduction in the thymic weight and cellularity involving different T cell maturation stages, mainly CD4 - CD8 - and CD4 + CD8 + thymocytes, as well as an increased presence of apoptotic cells, leading to significant thymic cortex reduction. Thymus atrophy showed no association with elevated serum cytokines levels, although increased glucocorticoid levels did. The severity of thymic damage in both models reached the same extend although it occurs at different stages of infection, showing that thymic atrophy does not depend on parasitemia level but on the specific host-parasite interaction., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 European Federation of Immunological Societies. Published by Elsevier B.V. All rights reserved.)

Drug therapy for leishmaniasis remains a major challenge as currently available drugs have limited efficacy, induce serious side-effects and are not accessible to everyone. Thus, the discovery of affordable drugs is urgently needed. Chalcones present a great potential as bioactive agents due to simple structure and functionalization capacity. The antileishmanial activity of different natural and synthetic chalcones have been reported. Here we report the synthesis of twenty-five novel prenylated chalcones that displayed antiparasitic activity in Leishmania mexicana. All the chalcones were evaluated at 5 µg/mL and eleven compounds exhibited a metabolic inhibition close to or exceeding 50%. Compounds 49, 30 and 55 were the three most active with IC 50 values < 10 μM. These chalcones also showed the highest selectivity index (SI) values. Interestingly 49 and 55 possessing a substituent at a meta position in the B ring suggests that the substitution pattern influences antileishmanial activity. Additionally, a tridimensional model of fumarate reductase of L. mexicana was obtained by homology modeling. Docking studies suggest that prenylated chalcones could modulate fumarate reductase activity by binding with good affinity to two binding sites that are critical for the target. In conclusion, the novel prenylated chalcones could be considered as promising antileishmanial agents., (© 2022. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Flow cytometry (FC) is widely used in microbiology, immunology, hematology, and oncology. In the veterinary field, FC enabled the study of the immune response in cattle infected with different pathogens, as well as vaccine testing. However, few fluorochrome-conjugated antibodies recognize bovine antigens, limiting the possible benefits of FC and the implementation of multiparametric analysis for more complex studies. Two cytometry panels with five colors each were designed and implemented for the study and identification of populations and subpopulations of T cells derived from the peripheral blood mononuclear cells of dairy heifers. Both panels detected differences in T cell subpopulations between heifers positively and negatively tested for tuberculin; they detected overexpression of CD25 + and CD45RO + in tuberculin-positive heifers after stimulation with a culture filtrate protein extract (CFPE) from Mycobacterium bovis ( M. bovis ). We identified subpopulations of T cells from peripheral blood mononuclear cells using two multicolor panels. These panels could be used to analyze total bovine blood in immunopathogenic studies and vaccine development. The same strategy could be implemented in other species of veterinary interest.

Leishmaniasis is a neglected vector-borne disease; there are different manifestations of the diseases and species involved, and cutaneous leishmaniasis caused by Leishmania (L.) mexicana is the most prevalent in Mexico. Currently, the drugs available for the treatment of leishmaniasis are toxic, expensive, and often ineffective; therefore, it is imperative to carry out research and development of new therapeutic alternatives, with natural products being an attractive option. In particular, oregano is a plant with worldwide distribution; in Mexico, two species: Lippia berlandieri Schauer and Poliomintha longiflora Gray are endemic. Both essential oils (EO's) have been reported to have antimicrobial activity attributed to their main components, thymol and carvacrol. In this research, the leishmanicidal effect and mechanism of cell death induced by L. berlandieri EO, P. longiflora EO, thymol, and carvacrol in L. mexicana promastigotes were determined in vitro. Additionally, the cytotoxic activity in mammalian cells was evaluated. L. berlandieri EO presented higher leishmanicidal activity (IC 50 = 41.78 µg/mL) than P. longiflora EO (IC 50 = 77.90 µg/mL). Thymol and carvacrol were the major components of both Mexican oregano EO's. Thymol presented higher leishmanial inhibitory activity (IC 50 = 22.39 µg/mL), above that of carvacrol (IC 50 = 61.52 µg/mL). All the EO's and compounds evaluated presented lower cytotoxic activity than the reference drug; thymol was the compound with the best selectivity index (SI). In all cases, apoptosis was identified as the main mechanism of death induced in the parasites. The leishmanicidal capacity of the Mexican oregano EO is an accessible and affordable alternative that can be further explored.

Cerebral malaria (CM) is a neurological complication derived from the Plasmodium falciparum infection in humans. The mechanisms involved in the disease progression are still not fully understood, but both the sequestration of infected red blood cells (iRBC) and leukocytes and an exacerbated host inflammatory immune response are significant factors. In this study, we investigated the effect of Monocyte Locomotion Inhibitory Factor (MLIF), an anti-inflammatory peptide, in a well-characterized murine model of CM. Our data showed that the administration of MLIF increased the survival and avoided the neurological signs of CM in Plasmodium berghei ANKA (PbA) infected C57BL/6 mice. MLIF administration down-regulated systemic inflammatory mediators such as IFN-γ, TNF-α, IL-6, CXCL2, and CCL2, as well as the in situ expression of TNF-α in the brain. In the same way, MLIF reduced the expression of CD31, CD36, CD54, and CD106 in the cerebral endothelium of infected animals and prevented the sequestration of iRBC and leucocytes in the brain microvasculature. Furthermore, MLIF inhibited the activation of astrocytes and microglia and preserved the integrity of the blood-brain barrier (BBB). In conclusion, our results demonstrated that the administration of MLIF increased survival and conferred neuroprotection by decreasing neuroinflammation in murine CM., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The biological activity of essential oils and their major components is well documented. Essential oils such as oregano and cinnamon are known for their effect against bacteria, fungi, and even viruses. The mechanism of action is proposed to be related to membrane and external cell structures, including cell walls. This study aimed to evaluate the biological activity of seven essential oils and eight of their major components against Gram-negative and Gram-positive bacteria, filamentous fungi, and protozoans. The antimicrobial activity was evaluated by determination of the Minimal Inhibitory Concentration for Bacillus cereus , Staphylococcus aureus , Listeria monocytogenes , Escherichia coli , Salmonella Typhimurium, Shigella sonnei , Aspergillus niger , Aspergillus ochraceus , Alternaria alternata, and Fusarium oxysporium , the half-maximal inhibitory concentration (IC 50 ) for Trypanosoma cruzi and Leishmania mexicana , and the median lethal dose (LD 50 ) for Giardia lamblia . Results showed that oregano essential oil showed the best antibacterial activity (66-100 µg/mL), while cinnamon essential oil had the best fungicidal activity (66-116 µg/mL), and both showed excellent antiprotozoal activity (22-108 µg/mL). Regarding the major components, thymol and carvacrol were also good antimicrobials (23-200 µg/mL), and cinnamaldehyde was an antifungal compound (41-75 µg/mL). The major components were grouped according to their chemical structure as phenylpropanoids, terpenoids, and terpinenes. The statistical analysis of the grouped data demonstrated that protozoans were more susceptible to the essential oils, followed by fungi, Gram-positive bacteria, and Gram-negative bacteria. The analysis for the major components showed that the most resistant microbial group was fungi, which was followed by bacteria, and protozoans were also more susceptible. Principal Component Analysis for the essential oils demonstrated the relationship between the biological activity and the microbial group tested, with the first three components explaining 94.3% of the data variability. The chemical structure of the major components was also related to the biological activity presented against the microbial groups tested, where the three first principal components accounted for 91.9% of the variability. The external structures and the characteristics of the cell membranes in the different microbial groups are determinant for their susceptibility to essential oils and their major components.

Dengue is a worldwide expanding threat caused by dengue virus (DENV) infection. To date, no specific treatment or effective vaccine is available. Antibodies produced by plasma cells (PCs) might be involved concomitantly in protection and severe dengue immunopathology. Although a massive appearance of PCs has been reported during acute DENV infection in humans, this response has been poorly characterized. Here, we show the dynamic of PC generation in immune-competent mice cutaneously inoculated with DENV compared with two control experimental groups: mice inoculated with inactivated DENV or with PBS. We found that PC numbers increased significantly in the skin-draining lymph node (DLN), peaking at day 10 and abruptly decreasing by day 14 after DENV inoculation. Class-switched IgG + PCs appeared from day 7 and dominated the response, while in contrast, the frequency of IgM + PCs decreased from day 7 onwards. Even though the kinetic of the response was similar between DENV- and iDENV-inoculated mice, the intensity of the response was significantly different. Interestingly, we demonstrated a similar PC response to virus antigens (E and prM) by ELISPOT. In situ characterization showed that PCs were distributed in the medullary cords and in close proximity to germinal centers (GCs), suggesting both an extrafollicular and a GC origin. Proliferating PCs (Ki-67 + ) were found as early as 3-day postinoculation, and in-depth analysis showed that these PCs were in active phases of cell cycle during the kinetic. Finally, we found a progressive appearance of high-affinity neutralizing DENV-specific IgG further supporting GC involvement. Of note, these antibodies seem to be highly cross-reactive, as a large proportion recognizes Zika virus (ZIKV). The strong PC response to skin-inoculated DENV in this work resembles the findings already described in humans. We consider that this study contributes to the understanding of the in vivo biology of the humoral immune response to DENV in an immunocompetent murine model., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this paper., (Copyright © 2021 Raúl A. Maqueda-Alfaro et al.)

Through the years, helminths have co-existed with many species. This process has allowed parasites to live within them for long periods and, in some cases, to generate offspring. In particular, this ability has allowed Fasciola hepatica to survive the diverse immunological responses faced within its wide range of hosts. The vast repertoire of molecules that are constantly secreted in large quantities by the parasite, acts directly on several cells of the immune system affecting their antiparasitic capacities. Interestingly, these molecules can direct the host immune response to an anti-inflammatory and regulatory phenotype that assures the survival of the parasite with less harm to the host. Based on these observations, some of the products of F. hepatica, as well as those of other helminths, have been studied, either as a total extract, extracellular vesicles or as purified molecules, to establish and characterize their anti-inflammatory mechanisms. Until now, the results obtained encourage further research directed to discover new helminth-derived alternatives to replace current therapies, which can be useful for people suffering from inflammatory diseases like autoimmunity or allergy processes that affect their life quality. In this review, some of the most studied molecules derived from F. hepatica and their modulating capacities are discussed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Dengue is a global health problem without current specific treatment nor safe vaccines available. While severe dengue is related to pre-existing non-neutralizing dengue virus (DENV) antibodies, the role of T cells in protection or pathology is unclear. Using cutaneous DENV infection in immunocompetent mice we previously showed the generation of PNA+ germinal centers (GCs), now we assessed the activation and proliferation of B and T cells in draining lymph nodes (DLNs). We found a drastic remodelling of DLN compartments from 7 to 14 days post-infection (dpi) with greatly enlarged B cell follicles, occupying almost half of the DLN area compared to ~24% in naïve conditions. Enormous clusters of proliferating (Ki-67+) cells inside B follicles were found 14 dpi, representing ~33% of B cells in DLNs but only ~2% in non-infected mice. Inside GCs, we noticed an important recruitment of tingle body macrophages removing apoptotic cells. In contrast, the percentage of paracortex area and total T cells decreased by 14-16 dpi, compared to controls. Scattered randomly distributed Ki-67+ T cells were found, similar to non-infected mice. CD69 expression by CD4+ and CD8+ T cells was minor, while it was remarkable in B cells, representing 1764.7% of change from basal levels 3 dpi. The apparent lack of T cell responses cannot be attributed to apoptosis since no significant differences were observed compared to non-infected mice. This study shows massive B cell activation and proliferation in DLNs upon DENV infection. In contrast, we found very poor, almost absent CD4+ and CD8+ T cell responses.

Salmonella enterica infections remain a challenging health issue, causing significant morbidity and mortality worldwide. Current vaccines against typhoid fever display moderate efficacy whilst no licensed vaccines are available for paratyphoid fever or invasive non-typhoidal salmonellosis. Therefore, there is an urgent need to develop high efficacy broad-spectrum vaccines that can protect against typhoidal and non-typhoidal Salmonella . The Salmonella outer membrane porins OmpC and OmpF, have been shown to be highly immunogenic antigens, efficiently eliciting protective antibody, and cellular immunity. Furthermore, enterobacterial porins, particularly the OmpC, have a high degree of homology in terms of sequence and structure, thus making them a suitable vaccine candidate. However, the degree of the amino acid conservation of OmpC among typhoidal and non-typhoidal Salmonella serovars is currently unknown. Here we used a bioinformatical analysis to classify the typhoidal and non-typhoidal Salmonella OmpC amino acid sequences into different clades independently of their serological classification. Further, our analysis determined that the porin OmpC contains various amino acid sequences that are highly conserved among both typhoidal and non-typhoidal Salmonella serovars. Critically, some of these highly conserved sequences were located in the transmembrane β-sheet within the porin β-barrel and have immunogenic potential for binding to MHC-II molecules, making them suitable candidates for a broad-spectrum Salmonella vaccine. Collectively, these findings suggest that these highly conserved sequences may be used for the rational design of an effective broad-spectrum vaccine against Salmonella ., (Copyright © 2020 Valero-Pacheco, Blight, Aldapa-Vega, Kemlo, Pérez-Toledo, Wong-Baeza, Kurioka, Perez-Shibayama, Gil-Cruz, Sánchez-Torres, Pastelin-Palacios, Isibasi, Reyes-Sandoval, Klenerman and López-Macías.)

Ursolic and oleanolic acids are natural isomeric triterpenes known for their anticancer activity. Here, we investigated the effect of triterpenes on the viability of A549 human lung cancer cells and the role of autophagy in their activity. The induction of autophagy, the mitochondrial changes and signaling pathway stimulated by triterpenes were systematically explored by confocal microscopy and western blotting. Ursolic and oleanolic acids induce autophagy in A549 cells. Ursolic acid activates AKT/mTOR pathways and oleanolic acid triggers a pathway independent on AKT. Both acids promote many mitochondrial changes, suggesting that mitochondria are targets of autophagy in a process known as mitophagy. The PINK1/Parkin axis is a pathway usually associated with mitophagy, however, the mitophagy induced by ursolic or oleanolic acid is just dependent on PINK1. Moreover, both acids induce an ROS production. The blockage of autophagy with wortmannin is responsible for a decrease of mitochondrial membrane potential (Δψ) and cell death. The wortmannin treatment causes an over-increase of p62 and Nrf2 proteins promote a detoxifying effect to rescue cells from the death conducted by ROS. In conclusion, the mitophagy and p62 protein play an important function as a survival mechanism in A549 cells and could be target to therapeutic control.

The available drugs for treating Leishmaniasis and American trypanosomiasis have high toxicity and multiple side effects, among other problems. More effective and less toxic treatments are urgently needed. A series of chalcones that contained a prenyloxy or geranyloxy substituent was synthesized and characterized. Each substituent was attached to the A ring in some compounds and to the B ring in others, with additional substituents placed on the chalcone moiety. The present aim was to evaluate the effect of the substitution pattern on leishmanicidal and trypanocidal activity. When tested at a single concentration, the compounds exerting a metabolic inhibition close to or exceeding 50% for Leishmania mexicana were 11, 17 and 12, and for Trypanosoma cruzi were 11, 17, 15 and 26. Upon determining the selectivity index (SI =IC 50 /CC 50 ), the values were 80.9, 1.24 and 55.12 for 11, 17 and 12 (respectively) versus L. mexicana, and 75.1, 1.43, 27.36 and 33.52 for 11, 17, 15 and 26 (respectively) versus T. cruzi. Structural isomers 11 and 17 showed activity for both the L. mexicana and T. cruzi strains, though the greater cytotoxic activity of 17 led to a lower SI. Compounds 12, 15 and 26 were species specific. For T. cruzi, the SI was higher for 11, 15 and 26 than for the reference drugs nifurtimox and benznidazole. The examination of promastigote morphology after exposing L. mexicana and T. cruzi to 11 revealed a decrease in cell density. The current findings suggest that 11 could be a useful lead compound for further SAR studies., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Platelets are considered as significant players in innate and adaptive immune responses. The adhesion molecules they express, including P-selectin, CD40L, and CD42b, facilitate interactions with many cellular effectors. Upon interacting with a pathogen, platelets rapidly express and enhance their adhesion molecules, and secrete cytokines and chemokines. A similar phenomenon occurs after exposure of platelets to thrombin, an agonist extensively used for in vitro activation of these cells. It was recently reported that the dengue virus not only interacts with platelets but possibly infects them, which triggers an increased expression of adhesion molecule P-selectin as well as secretion of IL-1β. In the present study, surface molecules of platelets like CD40L, CD42b, CD62P, and MHC class I were evaluated at 4 h of interaction with dengue virus serotype 2 (DENV-2), finding that DENV-2 induced a sharp rise in the membrane expression of all these molecules. At 2 and 4 h of DENV-2 stimulation of platelets, a significantly greater secretion of soluble CD40L (sCD40L) was found (versus basal levels) as well as cytokines such as GM-CSF, IL-6, IL-8, IL-10, and TNF-α. Compared to basal, DENV-2 elicited more than two-fold increase in these cytokines. Compared to the thrombin-induced response, the level generated by DENV-2 was much higher for GM-CSF, IL-6, and TNF-α. All these events induced by DENV end up in conspicuous morphological changes observed in platelets by confocal microscopy and transmission electron microscopy, very different from those elicited by thrombin in a more physiological scenery.

Background: In order to develop new larvicidal agents derived from phytochemicals, the larvicidal activity of fifty molecules that are constituent of essential oils was evaluated against Culex quinquefasciatus Say. Terpenes, terpenoids and phenylpropanoids molecules were included in the in vitro evaluation, and QSAR models using genetic algorithms were built to identify molecular and structural properties of biological interest. Further, to obtain structural details on the possible mechanism of action, selected compounds were submitted to docking studies on sterol carrier protein-2 (SCP-2) as possible target., Results: Results showed high larvicidal activity of carvacrol and thymol on the third and fourth larval stage with a median lethal concentration (LC 50 ) of 5.5 and 11.1 µg/mL respectively. Myrcene and carvacrol were highly toxic for pupae, with LC 50 values of 31.8 and 53.2 µg/mL. Structure-activity models showed that the structural property π-bonds is the largest contributor of larvicidal activity while ketone groups should be avoided. Similarly, property-activity models attributed to the molecular descriptor LogP the most contribution to larvicidal activity, followed by the absolute total charge (Qtot) and molar refractivity (AMR). The models were statistically significant; thus the information contributes to the design of new larvicidal agents. Docking studies show that all molecules tested have the ability to interact with the SCP-2 protein, wherein α-humulene and β-caryophyllene were the compounds with higher binding energy., Conclusions: The description of the molecular properties and the structural characteristics responsible for larvicidal activity of the tested compounds were used for the development of mathematical models of structure-activity relationship. The identification of molecular and structural descriptors, as well as studies of molecular docking on the SCP-2 protein, provide insight on the mechanism of action of the active molecules, and the information can be used for the design of new structures for synthesis as potential new larvicidal agents.

The larvicidal activity of essential oils cinnamon ( Cinnamomum verum J. Presl), Mexican lime ( Citrus aurantifolia Swingle) cumin ( Cuminum cyminum Linnaeus), clove ( Syzygium aromaticum (L.) Merr. & L.M.Perry), laurel ( Laurus nobilis Linnaeus), Mexican oregano ( Lippia berlandieri Schauer) and anise ( Pimpinella anisum Linnaeus)) and their major components are tested against larvae and pupae of Culex quinquefasciatus Say. Third instar larvae and pupae are used for determination of lethality and mortality. Essential oils with more than 90% mortality after a 30-min treatment are evaluated at different time intervals. Of the essential oils tested, anise and Mexican oregano are effective against larvae, with a median lethal concentration (LC 50 ) of 4.7 and 6.5 µg/mL, respectively. Anise essential oil and t -anethole are effective against pupae, with LC 50 values of 102 and 48.7 µg/mL, respectively. Oregano essential oil and carvacrol also have relevant activities. A kinetic analysis of the larvicidal activity, the oviposition deterrent effect and assays of the effects of the binary mixtures of chemical components are undertaken. Results show that anethole has synergistic effects with other constituents. This same effect is observed for carvacrol and thymol. Limonene shows antagonistic effect with β-pinene. The high larvicidal and pupaecidal activities of essential oils and its components demonstrate that they can be potential substitutes for chemical compounds used in mosquitoes control programs., Competing Interests: The authors declare that they have no conflict of interest.

Leishmaniasis is a neglected tropical disease caused by the parasite of the genus Leishmania. About 13 million people are infected worldwide, and it is estimated that 350 million are at risk of infection. Clinical manifestations depend on the parasite species and factors related to the host such as the immune system, nutrition, housing, and financial resources. Available treatments have severe side effects; therefore, research currently focuses on finding more active and less toxic compounds. Quinoxalines have been described as promising alternatives. In this context, 17 isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives were evaluated as potential leishmanicidal agents. Their effect on the cell metabolism of Leishmania mexicana promastigotes and their cytotoxic effects on the J774.A1 cell line and on erythrocytes were evaluated, and their selectivity index was calculated. Compounds T-069 (IC 50  = 1.49 μg/mL), T-070 (IC 50  = 1.71 μg/mL), T-072 (IC 50  = 6.62 μg/mL), T-073 (IC 50  = 1.25 μg/mL), T-085 (IC 50  = 0.74 μg/mL), and T-116 (IC 50  = 0.88 μg/mL) were the most active against L. mexicana promastigotes and their mechanism of action was characterized by flow cytometry and microscopy. Compound T-073, the most selective quinoxaline derivative, induced cell membrane damage, phosphatidylserine exposition, reactive oxygen species production, disruption of the mitochondrion membrane potential, and DNA fragmentation, all in a dose-dependent manner, indicating the induction of regulated necrosis. Light and transmission electron microscopy showed the drastic morphological changes induced and the mitochondrion as the most sensitive organelle in response to T-073. This study describes the mechanism by which active isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide quinoxalines affect the parasite.

Quinoxalines display diverse and interesting pharmacological activities as antibacterial, antiviral, antiparasitic and anticancer agents. Particularly, their 1`4-di- N -oxide derivatives have proved to be cytotoxic agents that are active under hypoxic conditions as that of solid tumours. A new series of quinoxaline 1`4-di- N -oxide substitutes at 7-position with esters group were synthetized and characterized by infrared (IR), proton nuclear magnetic resonance ( 1 H-NMR), spectroscopy, and elemental analysis. Seventeen derivatives (M1-M3, E1-E8, P1-P3 and DR1-DR3) were selected and evaluated for antitumor activities using the NCI-60 human tumor cell lines screen. Results showed that E7, P3 and E6 were the most active compounds against the cell lines tested. Substitutions at 7-position with esters group not necessarily affect the biological activity, but the nature of the esters group could exert an influence on the selectivity. Additionally, substitutions at 2-position influenced the cytotoxic activity of the compounds.

More than half of the human population is exposed to mosquito-borne infections. Climate change and the emergence of strains resistant to traditionally used insecticides have motivated the search of new agents for mosquito population control. Essential oils have been effective repellents and larvicidal agents.The aim of this work was to review research studies conducted in recent years on the larvicidal activity of essential oils and their components against Aedes, Anopheles and Culex mosquitoes, as well as the latest reports about their possible mechanism of action.

Chagas disease or American trypanosomiasis is a worldwide public health problem. In this work, we evaluated 26 new propyl and isopropyl quinoxaline-7-carboxylate 1,4-di-N-oxide derivatives as potential trypanocidal agents. Additionally, molecular docking and enzymatic assays on trypanothione reductase (TR) were performed to provide a basis for their potential mechanism of action. Seven compounds showed better trypanocidal activity on epimastigotes than the reference drugs, and only four displayed activity on trypomastigotes; T-085 was the lead compound with an IC50 = 59.9 and 73.02 µM on NINOA and INC-5 strain, respectively. An in silico analysis proposed compound T-085 as a potential TR inhibitor with better affinity than the natural substrate. Enzymatic analysis revealed that T-085 inhibits parasite TR non-competitively. Compound T-085 carries a carbonyl, a CF3, and an isopropyl carboxylate group at 2-, 3- and 7-position, respectively. These results suggest the chemical structure of this compound as a good starting point for the design and synthesis of novel trypanocidal derivatives with higher TR inhibitory potency and lower toxicity.

Bovine lactoferrin (bLf) up-modulates intestinal IgA that is essential for homeostasis and which might confer protection to the distal small intestine that is vulnerable to inflammation. This study analyzed the effects of bLf administered orally on the IgA response at inductive (Peyer's patches) and effector (lamina propria) sites of the distal small intestine in mice. Groups of five healthy male BALB/c mice were orally treated with 5 mg of bLf for 7, 14, 21, or 28 days. Then, mice were killed and the distal small intestine was dissected. Intestinal fluid samples were analyzed to determine IgA and IgM levels by enzyme-immuno assay. Peyer's patches and lamina propria were analyzed for IgA(+) or IgM(+) plasma cells, B, CD4(+) T and CD8(+) T cells as well as CD4(+) T cells positive for either pro-inflammatory cytokines [tumor necrosis factor (TNF)-α, interferon-γ and interleukin (IL)-12] or for IgA-producing ILs (IL-4, -5, -10 and -6) by cytofluorometry. Antibodies, antibody-secreting cells, and B and T responses in both Peyer's patches and lamina propria were higher in bLf-treated than bLf-untreated mice. The generation of IL-10 and IL-6 CD4(+) T cells in Peyer's patches or TNF-α and IL-12 CD4(+) T cells in lamina propria showed similar response patterns. On days 14 and 28, cytokine/IL CD4(+) T cell responses were increased in Peyer's patches or decreased in lamina propria. The effect of bLf on the elicitation of IgA indicates a potential application of bLf as a nutraceutical to control inflammation in the distal small intestine.

Thiol reagents were shown to act as potent inhibitors of L5178-Y murine leukemia cell proliferation. A series of aryl maleimides (AMI) was synthesized and evaluated theoretically for global and local reactivity, showing their selectivity for thiol groups, due to a reaction of the vinyl moiety (a soft acid) with thiols (a soft base). Two AMI that are benzoic acid derivatives (1f and 1h) were tested with an in vitro and ex vivo model to evaluate their reactivity with thiols and their activity in L5178-Y cells. The in vitro reactions clearly showed a selective Michael type 1,4-addition reaction between thiols (glutathione and N-acetylcysteine, which are nucleophiles) and the AMI (1f and 1h, which are electrophiles). In cell cultures, the compounds induced a decreasing cellular viability and an apoptotic effect of up to 59.8% at 48 h. The ex vivo experimental model showed an important reduction of thiol levels in cells treated with 1h. Decreased cellular viability and increased apoptosis were confirmed by flow cytometry, DNA fragmentation and microscopy analysis (cytological studies). The increase in apoptosis on L5178-Y cells probably occurred, at least in part, by a decrease in glutathione levels and an increase in free radicals concentration. The decreased glutathione levels seem to make cancer cells more susceptible to death by apoptosis, and should certainly make them more vulnerable to a less aggressive treatment.

Background: Essential oils and their constituents are commonly known for their antibacterial, antifungal and antiparasitic activity, and there are also reports on the antimycobacterial properties, but more experimental data are needed for the description of the mechanism of action or structural (and molecular) properties related to the antimicrobial activity., Methods: Twenty-five constituents of essential oils were evaluated against Mycobacterium tuberculosis H37Rv and Mycobacterium bovis AN5 by the Alamar Blue technique. Twenty compounds were modeled using in silico techniques descriptor generation and subsequent QSAR model building using genetic algorithms. The p-cymene, menthol, carvacrol and thymol were studied at the quantum mechanical level through the mapping of HOMO and LUMO orbitals. The cytotoxic activity against macrophages (J774A) was also evaluated for these four compounds using the Alamar Blue technique., Results: All compounds tested showed to be active antimicrobials against M. tuberculosis. Carvacrol and thymol were the most active terpenes, with MIC values of 2.02 and 0.78 μg/mL respectively. Cinnamaldehyde and cinnamic acid were the most active phenylpropanes with MIC values of 3.12 and 8.16 μg/mL respectively. The QSAR models included the octanol-water partition (LogP) ratio as the molecular property that contributes the most to the antimycobacterial activity and the phenolic group (nArOH) as the major structural element., Conclusions: The description of the molecular properties and the structural characteristics responsible for antimycobacterial activity of the compounds tested, were used for the development of mathematical models that describe structure-activity relationship. The identification of molecular and structural descriptors provide insight into the mechanisms of action of the active molecules, and all this information can be used for the design of new structures that could be synthetized as potential new antimycobacterial agents.

Neutrophils are one the earliest, crucial innate defenses against innumerable pathogens. Their main microbicidal activities include phagocytosis and degranulation, with many pharmacologically active molecules contributing to inflammation. Recently, a novel antimicrobial mechanism was discovered; the Neutrophil Extracelullar Traps (NETs) formed by extrusion of DNA and associated molecules (histones, elastase, antimicrobial peptides, among others) which trap and kill microorganisms. Since NETs were recently described, research has focused on their induction and microbicidal properties, and recently on disease involvement. However, the functional consequences of NETs interacting with other immune cells, either resident or recruited during early inflammation, have not been assessed. We therefore investigated the consequences of exposing two major APCs, macrophages (Mfs) and conventional Dendritic Cells (cDCs) to NETs. Our data revealed that at early times (30 min), both Antigen Presenting Cells (APCs) showed induction of important costimulatory molecules (CD80, CD86). Unexpectedly, however, at later times (6 and 24 hours) NETs apparently triggered a cell death process in these APCs by a caspase- and Apoptosis induced factor (AIF)-dependent pathway, suggesting mitochondrial damage. By rhodamine-123 labelling we found that in both APCs, relatively prolonged exposure to NETs or their components importantly decreased the mitochondrial membrane potential. Ultrastructural analysis confirmed mitochondrial alterations in both APCs. Our results would suggest that early in inflammation, NETs can activate the two main APCs (Mfs and cDCs), but as the process continues, NETs can then initiate apoptosis of these cells through mitochondrial harm. Conceivable, this "late" induction of cell death in these two APCs might start limiting an ongoing inflammatory process to control it.

Colonization of epithelium by microorganisms leads to inflammatory responses. In some cases an anti-apoptotic response involving the cellular inhibitor of apoptosis protein-2 (cIAP-2) also occurs. Although strong expression of cIAP-2 has been observed in lesional skin from psoriatic patients and in HaCaT keratinocytes treated with peptidoglycan (PGN) from Staphylococcus aureus, anti-apoptotic responses induced in the skin by cIAP-2 have seldom been studied. In this study, the effect of PGN on TNF-α-induced apoptotic HaCaT keratinocytes was assessed. Morphological analysis, quantification of cells with DNA fragmentation and active caspase-3 detection was performed to assess apoptotic cell death. Greater LL-37 and cIAP-2 production was found in keratinocytes stimulated with PGN than in non-treated cells (P < 0.05). In comparison with cells treated with TNF-α only, a significant reduction in apoptotic cell death was observed when HaCaT were pretreated with PGN before inducing apoptosis with TNF-α (P < 0.05). In addition, an inhibitor of cIAP-2 activity (LCL161) stopped the PGN effect. These findings show that PGN from S. aureus has an anti-apoptotic effect in keratinocytes mediated by cIAP-2 production, suggesting that this anti-apoptotic activity could favor proliferation of keratinocytes in psoriasis., (© 2014 The Societies and Wiley Publishing Asia Pty Ltd.)

PD-L1 expression plays a critical role in the impairment of T cell responses during chronic infections; however, the expression of PD-L1 on T cells during acute viral infections, particularly during the pandemic influenza virus (A(H1N1)pdm09), and its effects on the T cell response have not been widely explored. We found that A(H1N1)pdm09 virus induced PD-L1 expression on human dendritic cells (DCs) and T cells, as well as PD-1 expression on T cells. PD-L1 expression impaired the T cell response against A(H1N1)pdm09 by promoting CD8⁺ T cell death and reducing cytokine production. Furthermore, we found increased PD-L1 expression on DCs and T cells from influenza-infected patients from the first and second 2009 pandemic waves in Mexico City. PD-L1 expression on CD8⁺ T cells correlated inversely with T cell proportions in patients infected with A(H1N1)pdm09. Therefore, PD-L1 expression on DCs and T cells could be associated with an impaired T cell response during acute infection with A(H1N1)pdm09 virus.

Intestinal homeostasis effectors, secretory IgA (SIgA) and polymeric immunoglobulin receptor (pIgR), have been evaluated in proximal and distal small intestine with moderate-exercise training but not with strenuous exercise or a combination of these two protocols. Therefore, two groups of mice (n=6-8) were submitted to strenuous exercise, one with and one without previous training. The control group had no exercise protocol. Assessment was made of intestinal SIgA and plasma adrenal hormones (by immunoenzymatic assay), alpha-chain and pIgR proteins in intestinal mucosa (by Western blot), lamina propria IgA plasma-cells (by cytofluorometry), mRNA expression (by real-time PCR) for pIgR, alpha- and J-chains in liver and intestinal mucosa, and pro- and anti-inflammatory cytokines in mucosa samples. Compared to other exercise protocols, training plus strenuous exercise elicited: (1) higher levels of SIgA and pIgR in the proximal intestine (probably by hepatobiliary contribution); (2) higher levels of SIgA in the distal segment; (3) lower mRNA expression of some SIgA- and most pro-inflammatory pIgR-producing cytokines. SIgA and pIgR in both segments were derived from an existing pool of their corresponding producing cells. The apparent decreased translation of mRNA transcripts underlies lower levels of SIgA and pIgR in distal than proximal small intestine. There was no significant difference in the relatively high adrenal hormone levels found in both exercised groups. Further study is required about the effects of training plus strenuous exercise on pool-derived SIgA levels and mRNA expression of pro-inflammatory pIgR-producing cytokines. These results could have important implications for intestinal disorders involving inflammation and infection., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Recently it was discovered that neutrophils can generate extracellular fibers called NET (neutrophil extracellular traps), which are composed of a skeleton of DNA “decorated” with many cytoplasmic –including enzymes– and nuclear components.The NET are a physical barrier that prevent the spread of microorganisms and facilitate the cell death by promoting a high local concentration of antimicrobial molecules. On the other hand, the fibrous structure limits the damage to the tissue where they are generated by restricting the range of molecules that are released by the neutrophil. This paper describes this new form of cell death and the implications this may have on different diseases.

The effects of stress on the mucosal immune responses in inflammatory disorders of the gut, as well as on salivary and intestinal IgA levels are well known. However, its effects on the structure and function of the NALT have not yet been reported, and are examined in the present study. Balb/c mice were submitted to restraint stress for 3h per day during 4 or 8d. The immunohistochemistry and flow cytometric analysis revealed that repeated restraint stress (4 and 8d) decreased the percentage, compared to the control group, of CD3(+) and CD4(+) T cells, without affecting the percentage of CD8(+) T cells or B220(+) cells (B cells). The numbers of IELs (CD4(+) and CD8(+) T cells) were lower at 4d of stress and higher at 8d. IgA(+) cells in NALT and nasal IgA levels showed a similar pattern, being significantly lower at 4d of stress and significantly higher at 8d. In summary, repeated restraint stress altered the distribution and number of lymphocytes and IgA(+) cells in nasal mucosa, probably due to changes in norepinephrine and corticosterone levels., (Copyright © 2010 Elsevier B.V. All rights reserved.)

The few reports that have analyzed the effects of stress on the immune cells of the intestinal mucosa or the functions of these cells have tended to focus on S-IgA levels in saliva, and these studies have shown contradictory results. The principal objective of this study was to analyze the effects of repeated restraint stress on the number and distribution of immune cells in Peyer's patches (PPs) as well as the effects of glucocorticoid and catecholamine administration on the same stress-related parameters. Upon analyzing the effect of repeated restraint stress on PPs, it was found that there was no modification in the morphological structure of the PPs but that restraint stress reduced the total number of lymphocytes and the number of CD8+ T cells, B cells, and plasma cells in PPs. Only at the site of PPs where IgA-producing plasma cells are most numerous (the dome) was a decrease found in this type of cell. These effects were due at least in part to the effect of glucocorticoids and catecholamines. Since IgA produced in PPs is a natural antibody that impedes bacterial infections, repeated stress may favor the entry of pathogens through the intestine., (2011 S. Karger AG, Basel.)

In this study, we investigated the cytotoxic effect of perezone, a constituent isolated from the roots of Perezia spp. and of its synthetic isomer isoperezone on the K562 human leukemia cell line. Perezone showed greater cytotoxic effect than isoperezone but both compounds were found to induce cytotoxicity trough a caspase-dependent and a caspase-independent mechanisms; important changes in their light scattering properties, phosphatidylserine translocation and mitochondrial membrane potential disruption were detected by cytometry. The mechanism of death induction of each compound showed interesting concentration-dependent differences. Neither compound induced the apoptosis inducing factor., ((c) 2009 Elsevier GmbH. All rights reserved.)

Ectocytosis, the cellular process by which ectosomes (Ects) are released, is an important phenomenon by which eukaryotic cells exchange molecular information. Ects released from N-formylmethionyl-leucyl-phenylalanine (fMLP)-activated human polymorphonuclear neutrophils (PMNs) have recently been characterized. Molecules such as CD35 and phosphatidylserine (PS), and enzymes such as myeloperoxidase and elastase were found in these vesicles, suggesting that Ects from PMNs could function as ecto-organelles with anti-microbial activity. Here we show for the first time that human PMNs release ectosomes in response to Mycobacterium tuberculosis H37Rv infection. We found that the release of ectosomes was not associated exclusively with mycobacterial infection since infection with other microorganisms (e.g., Leishmania mexicana, Staphylococcus aureus, and Escherichia coli or activation with phorbol myristate acetate (PMA)) also induced ectocytosis. Ects release started as early as 10min after infection or activation. Expression of CD35, PS, Rab5, Rab7 and gp91(Phox), a subunit of Cyt b555 was demonstrated on the Ects membrane. Based on our observations we conclude that Ects are released from human neutrophils in response to cell activation and that this process is not related to apoptosis.

Compounds containing B-N bonds have shown interesting biological activity. One class of such molecules is the 2,2-diphenyl-1,3,2-oxazaborolidin-5-ones (3a-j), which contain a B-N bond, have an alpha-amino acid moiety in the heterocycle, and have an exocyclic moiety related to an amino acid. The purpose of this work was to determine the inhibitory effects of 3a-j on the proliferation of murine L5178Y lymphoma cells. A new five-membered heterocyclic nucleus with apoptotic activity was found. The target products showed potent cytotoxicity in the L5178Y cell line. Among them, 3a exhibited the highest antineoplastic activity in L5178Y cells with an IC(50) value of 22.5+/-0.2 microM.