Synthesis of Antiprotozoal 2-(4-Alkyloxyphenyl)-Imidazolines and Imidazoles and Their Evaluation on Leishmania mexicana and Trypanosoma cruzi.

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 MnO2 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 IC50 < 10 µg/mL. Among these compounds, 27-31 were the most active, with IC50 values < 1 µg/mL (similar to the reference drugs). In the evaluation on epimastigotes of T. cruzi, only 30 and 36 reached an IC50 < 1 µg/mL, showing better inhibition than both reference drugs. However, compounds 29, 33, and 35 also demonstrated attractive trypanocidal activities, with IC50 values < 10 µg/mL, similar to the values for benznidazole and nifurtimox.

Adenosine increases PD-L1 expression in mesenchymal stromal cells derived from cervical cancer through its interaction with A2AR/A2BR and the production of TGF-ß1.

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-ß1). In previous studies, we reported that adenosine (Ado), generated by the adenosinergic activity of cervical cancer (CeCa) cells, induces the production of TGF-ß1 by interacting with A2AR/A2BR. In the present study, we provide evidence that Ado induces the production of TGF-ß1 in MSCs derived from CeCa tumors (CeCa-MSCs) by interacting with both receptors and that TGF-ß1 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 A2AR and A2BR, respectively, or SB-505124, a selective TGF-ß1 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-ß1 to increase PD-L1 expression. Those results strongly suggest the presence of a feedback mechanism among the adenosinergic pathway, the production of TGF-ß1, 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.

Repositioning FDA-Approved Drug Against Chagas Disease and Cutaneous Leishmaniosis by Structure-Based Virtual Screening.

BACKGROUND: Chagas disease and cutaneous leishmaniasis, two parasitic diseases caused by Trypanosoma cruzi (T. cruzi) and Leishmania mexicana (L. mexicana), respectively, have a major global impact. Current pharmacological treatments for these diseases are limited and can cause severe side effects; thus, there is a need for new antiprotozoal drugs. METHODS: Using molecular docking, this work describes a structure-based virtual screening of an FDA-approved drug library against Trypanosoma cruzi and Leishmania mexicana glycolytic enzyme triosephosphate isomerase (TIM), which is highly conserved in these parasites. The selected compounds with potential dual inhibitory activity were tested in vitro to confirm their biological activity. RESULTS: The study showed that five compounds: nilotinib, chlorhexidine, protriptyline, cyproheptadine, and montelukast, were more active against T. cruzi, than the reference drugs, nifurtimox and benznidazole while chlorhexidine and protriptyline were the most active against L. mexicana. CONCLUSIONS: The analysis of these compounds and their structural characteristics may provide the basis for the development of new antiprotozoal agents.

Imipramine Administration in Brucella abortus 2308-Infected Mice Restores Hippocampal Serotonin Levels, Muscle Strength, and Mood, and Decreases Spleen CFU Count.

Brucellosis infection causes non-specific symptoms such as fever, chills, sweating, headaches, myalgia, arthralgia, anorexia, fatigue, and mood disorders. In mouse models, it has been associated with increased levels of IL-6, TNF-α, and IFN-γ, a decrease in serotonin and dopamine levels within the hippocampus, induced loss of muscle strength and equilibrium, and increased anxiety and hopelessness. Imipramine (ImiP), a tricyclic antidepressant, is used to alleviate neuropathic pain. This study evaluated the effects of ImiP on Balb/c mice infected with Brucella abortus 2308 (Ba) at 14- and 28-days post-infection. Serum levels of six cytokines (IFN-γ, IL-6, TNF-α, IL-12, MCP-1. and IL-10) were assessed by FACS, while the number of bacteria in the spleen was measured via CFU. Serotonin levels in the hippocampus were analyzed via HPLC, and behavioral tests were conducted to assess strength, equilibrium, and mood. Our results showed that mice infected with Brucella abortus 2308 and treated with ImiP for six days (Im6Ba14) had significantly different outcomes compared to infected mice (Ba14) at day 14 post-infection. The mood was enhanced in the forced swimming test (FST) (p < 0.01), tail suspension test (TST) (p < 0.0001), and open-field test (p < 0.0001). Additionally, there was an increase in serotonin levels in the hippocampus (p < 0.001). Furthermore, there was an improvement in equilibrium (p < 0.0001) and muscle strength (p < 0.01). Lastly, there was a decrease in IL-6 levels (p < 0.05) and CFU count in the spleen (p < 0.0001). At 28 days, infected mice that received ImiP for 20 days (Im20Ba28) showed preservation of positive effects compared to infected mice (Ba28). These effects include the following: (1) improved FST (p < 0.0001) and TST (p < 0.0001); (2) better equilibrium (p < 0.0001) and muscle strength (p < 0.0001); (3) decreased IL-6 levels (p < 0.05); and (4) reduced CFU count in the spleen (p < 0.0001). These findings suggest the potential for ImiP to be used as an adjuvant treatment for the symptoms of brucellosis, which requires future studies.

Application of Multiparametric Flow Cytometry Panels to Study Lymphocyte Subpopulations in Tuberculin-Positive Cattle.

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.

Synthesis, characterization and evaluation of prenylated chalcones ethers as promising antileishmanial compounds.

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 IC50 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.

Irradiation and parasitism affect the ability of larval hemocytes of Anastrepha obliqua for phagocytosis and the production of reactive oxygen species.

The development of the parasitoid Doryctobracon crawfordi (Viereck) (Hymenoptera: Braconidae) in Anastrepha obliqua (McQuart) (Diptera: Tephritidae) larvae is unviable in nature; however, if the host larva is irradiated at 160 Gy, the parasitoid develops and emerges successfully. This suggests that radiation affects the immune responses of A. obliqua larvae, while the underlying mechanisms remain to be revealed. Using optical and electronic microscopies we determined the number and type of hemocyte populations found inside the A. obliqua larvae, either nonirradiated, irradiated at 160 Gy, parasitized by D. crawfordi, or irradiated and parasitized. Based on flow cytometry, the capacity to produce reactive oxygen species (ROS) was determined by the 123-dihydrorhodamine method in those hemocyte cells. Five cell populations were found in the hemolymph of A. obliqua larvae, two of which (granulocytes and plasmatocytes) can phagocytize and produce ROS. A reduction in the number of cells, mainly of the phagocytic type, was observed, as well as the capacity of these cells to produce ROS, when A. obliqua larvae were irradiated. Both radiation and parasitization decreased the ROS production, and when A. obliqua larvae were irradiated followed by parasitization by D. crawfordi, the reduction of the ROS level was even greater. In contrast, a slight increase in the size of these cells was observed in the hemolymph of the parasitized larvae compared to those in nonparasitized larvae. These results suggest that radiation significantly affects the phagocytic cells of A. obliqua and thus permits the development of the parasitoid D. crawfordi.

Mexican Oregano (Lippia berlandieri Schauer and Poliomintha longiflora Gray) Essential Oils Induce Cell Death by Apoptosis in Leishmania (Leishmania) mexicana Promastigotes.

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 (IC50 = 41.78 µg/mL) than P. longiflora EO (IC50 = 77.90 µg/mL). Thymol and carvacrol were the major components of both Mexican oregano EO's. Thymol presented higher leishmanial inhibitory activity (IC50 = 22.39 µg/mL), above that of carvacrol (IC50 = 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.

Low Concentration of the Neutrophil Proteases Cathepsin G, Cathepsin B, Proteinase-3 and Metalloproteinase-9 Induce Biofilm Formation in Non-Biofilm-Forming Staphylococcus epidermidis Isolates.

Neutrophils play a crucial role in eliminating bacteria that invade the human body; however, cathepsin G can induce biofilm formation in a non-biofilm-forming Staphylococcus epidermidis 1457 strain, suggesting that neutrophil proteases may be involved in biofilm formation. Cathepsin G, cathepsin B, proteinase-3, and metalloproteinase-9 (MMP-9) from neutrophils were tested on the biofilm induction in commensal (skin isolated) and clinical non-biofilm-forming S. epidermidis isolates. From 81 isolates, 53 (74%) were aap+, icaA−, icaD− genotype, and without the capacity of biofilm formation under conditions of 1% glucose, 4% ethanol or 4% NaCl, but these 53 non-biofilm-forming isolates induced biofilm by the use of different neutrophil proteases. Of these, 62.3% induced biofilm with proteinase-3, 15% with cathepsin G, 10% with cathepsin B and 5% with MMP -9, where most of the protease-induced biofilm isolates were commensal strains (skin). In the biofilm formation kinetics analysis, the addition of phenylmethylsulfonyl fluoride (PMSF; a proteinase-3 inhibitor) showed that proteinase-3 participates in the cell aggregation stage of biofilm formation. A biofilm induced with proteinase-3 and DNAse-treated significantly reduced biofilm formation at an early time (initial adhesion stage of biofilm formation) compared to untreated proteinase-3-induced biofilm (p < 0.05). A catheter inoculated with a commensal (skin) non-biofilm-forming S. epidermidis isolate treated with proteinase-3 and another one without the enzyme were inserted into the back of a mouse. After 7 days of incubation period, the catheters were recovered and the number of grown bacteria was quantified, finding a higher amount of adhered proteinase-3-treated bacteria in the catheter than non-proteinase-3-treated bacteria (p < 0.05). Commensal non-biofilm-forming S. epidermidis in the presence of neutrophil cells significantly induced the biofilm formation when multiplicity of infection (MOI) 1:0.01 (neutrophil:bacteria) was used, but the addition of a cocktail of protease inhibitors impeded biofilm formation. A neutrophil:bacteria assay did not induce neutrophil extracellular traps (NETs). Our results suggest that neutrophils, in the presence of commensal non-biofilm-forming S. epidermidis, do not generate NETs formation. The effect of neutrophils is the production of proteases, and proteinase-3 releases bacterial DNA at the initial adhesion, favoring cell aggregation and subsequently leading to biofilm formation.

Relevance of Helminth-Microbiota interplay in the host immune response.

Helminths have been present throughout the evolution of humans, promoting the development and maturation of the host immune system. However, this interaction is not exclusive between these two organisms, as the microbiota is also involved in this human evolutionary process and maintains a balanced relationship inside the host. Consequently, helminths have been forced to interact and co-evolve with the microbiota, shaping microbial communities and allowing the development of reciprocal mechanisms that favour their establishment. This helminth-microbiota association, in turn, induced the activation of different host immunoregulatory pathways to preserve health by preventing the development of some diseases associated with inflammatory immune responses. Unfortunately, this collaborative relationship can be quali- and quantitatively altered by the diet and the use of antibacterial and antihelminthic drugs. Understanding the mechanisms involved in this complex three-way communication that has continued for many years is crucial for preserving health and for the generation of new therapeutic alternatives.

Differential Antimicrobial Effect of Essential Oils and Their Main Components: Insights Based on the Cell Membrane and External Structure.

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 (IC50) for Trypanosoma cruzi and Leishmania mexicana, and the median lethal dose (LD50) 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.

Robust Plasma Cell Response to Skin-Inoculated Dengue Virus in Mice.

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.

Fasciola hepatica-derived molecules as potential immunomodulators.

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.

Anti-Inflammatory and Antioxidant Activities of the Methanolic Extract of Cyrtocarpa procera Bark Reduces the Severity of Ulcerative Colitis in a Chemically Induced Colitis Model.

Cyrtocarpa procera is a plant used in traditional Mexican medicine to treat different gastrointestinal problems. Here, we investigated the effects of a C. procera methanolic extract in DSS-induced colitis mice. Ulcerative colitis (UC) was induced by administering 4% DSS in drinking water to female BALB/c mice. Compared to untreated mice with UC, the treatment group receiving the C. procera extract presented less severe UC symptoms of diarrhea, bleeding, and weight loss. Additionally, colon shortening was significantly reduced, and at the microscopic level, only minor damage was observed. Levels of proinflammatory cytokines such as TNF-α, IL-1ß, and IFNγ in serum as well as the MPO activity in the colon were significantly reduced in the C. procera methanolic extract-treated group. Moreover, the extract of C. procera reduced oxidative stress during UC, preventing the deterioration of the activity of antioxidant enzymes such as SOD, CAT, and GPx. Additionally, the extract decreased lipid peroxidation damage and its final products, such as malondialdehyde (MDA). In agreement with this, in vitro assays with the C. procera extract displayed good antioxidant capacity, probably due to the presence of polyphenolic compounds, in particular the flavonoids that were identified, such as chrysin, naringenin, kaempferol, and catechin, which have been reported to have anti-inflammatory and antioxidant activities. Therefore, the improvement of UC by the C. procera methanolic extract may be related to the action mechanisms of these compounds.

Cutaneous Dengue Virus Inoculation Triggers Strong B Cell Reactions but Contrastingly Poor T Cell Responses.

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.

Extracellular vesicles released by J774A.1 macrophages reduce the bacterial load in macrophages and in an experimental mouse model of tuberculosis.

BACKGROUND: 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.

Ursolic and Oleanolic Acids Induce Mitophagy in A549 Human Lung Cancer Cells.

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.

Novel prenyloxy chalcones as potential leishmanicidal and trypanocidal agents: Design, synthesis and evaluation.

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 =IC50/CC50), 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.

Conservation of the OmpC Porin Among Typhoidal and Non-Typhoidal Salmonella Serovars.

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 ß-sheet within the porin ß-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.

Dengue Virus Induces the Release of sCD40L and Changes in Levels of Membranal CD42b and CD40L Molecules in Human Platelets.

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-1ß. 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.