TLR9 activation is required for cytotoxic response elicited by baculovirus capsid display.

Although the baculovirus Autographa californica multiple nuclear polyhedrosis virus (AcMNPV) infects lepidopteran invertebrates as natural hosts, represents an efficient vector for vaccine development. Baculovirus surface display induces strong humoral responses against viruses and parasites. A novel strategy based on capsid display carrying foreign antigens in the AcMNPV particle further improved the immune response by eliciting CD8+ T cell activation. In this study, we analyze the intracellular mechanisms and signalling pathways involved in CD8+ T cell activation by capsid display. Our results show that baculovirus can attach to the cell surface, enter dendritic cells (DCs), transit within endocytic vesicles and escape to the cytosol for further degradation by the proteasome. We found that the availability of viral proteins, endosomal acidification, and proteasome activity are needed for efficient Major Histocompatibility Complex class-I presentation by baculovirus carrying Ovalbumin in the viral capsid. Importantly, we demonstrated with this strategy that the induction of cytotoxic T cells and IL-12 production by DCs are TLR9-dependent and STING-independent. Finally, our study shows differential intracellular processing for capsid and surface baculovirus proteins in DCs and highlights the role of different danger receptors during cytotoxic T cell priming through the capsid display delivery system, which could lead to improved baculovirus-based vaccines development.

Antibacterial effects of extracts obtained from plants of Argentina: Bioguided isolation of compounds from the anti-infectious medicinal plant Lepechinia meyenii.

ETHNOPHARMACOLOGICAL RELEVANCE: The mostly native species from Argentina are used in traditional medicine generally for the treatment of pain and inflammation, respiratory, gastro-intestinal and urinary disorders and as antiseptics. AIM OF THE STUDY: Since these ailments may be associated with bacterial infections and that it is necessary to discover alternative compounds with antibacterial activity, 69 extracts from these plants were screened for their activity against pathogenic bacteria. The most effective extract was then submitted to bioguided isolation to obtain the compounds responsible for this activity. MATERIALS AND METHODS: Extracts and fractions were screened using agar dilution, and compounds using microbroth dilution methods. A large panel of pathogenic bacteria was used, especially methicillin-susceptible Staphylococcus aureus (MSSA) and methicillin-resistant S. aureus (MRSA). Bioguided fractionation was performed using successive chromatographic techniques, while the chemical structures of the isolated compounds were determined by nuclear magnetic resonance (NMR). Additionally, a series of derivatives of the most active compound were prepared in order to study the chemical features required for achieving the antibacterial effect. RESULTS: Lepechinia meyenii (Walp.) Epling (Lamiaceae) extract showed itself the most effective, with minimum inhibitory concentration (MIC) against Gram positive and negative bacteria ranging from 62.5 to 500 µg/mL, and showing better activity on MRSA than on MSSA. Activity-guided fractionation yielded the abietanes carnosol (1), rosmanol (2) and carnosic acid (3) as active principles, with MICs ranging from 15.6-31.2, 15.6-62.5 and 7.8-15.6 µg/mL, respectively against 15 MRSA strains, and 15.6-31.2, 31.2-62.5 and 7.8-15.6 µg/mL, respectively against 11 MSSA strains, maintaining higher activity against the resistant bacteria, as does the extract. In addition, Enterococcus faecalis was sensitive to 1-3 with MICs of 15.6-62.5 µg/mL. The structure activity analysis showed that 12-OH is necessary for remarkable activity, but methylation in C-20 significantly increased this, as observed with 20-methyl carnosate (5) displaying the greatest effect, even more so than 3, with MICs of 3.9 µg/mL against all the tested MRSA and 3.9-7.8 µg/mL against the MSSA. CONCLUSIONS: The results of this study contribute to validate the traditional antibacterial use of species native to Argentina, particularly of L. meyenii. The chemical structures of the compounds obtained may aid the design of antibacterial agents, especially those effective against MRSA.

Inhibitory effects of compounds isolated from Lepechinia meyenii on tyrosinase.

To contribute enzymatic browning inhibitors to the food industry and also extend knowledge about the phytochemical profile of the anti-tyrosinase plant Lepechinia meyenii, its ethanol extract was subjected to bioguided fractionation. Three hydroxycinnamic acids, p-coumaric acid (1), caffeic acid (2) and rosmarinic acid (3), were isolated as mainly responsible for its activity. Compounds 1, 2 and 3 showed themselves highly effective for inhibiting tyrosinase with IC50 values of 0.30, 1.50 and 4.14 µM, respectively, for monophenolase activity and 0.62, 2.30 and 8.59 µM, respectively for diphenolase activity. This is the first report describing the isolation of the compounds causing the tyrosinase inhibitory activity of L. meyenii extract. The inhibitory kinetics of 1-3 using both L-tyrosine and L-DOPA as substrates was investigated and the results obtained were discussed at molecular level by docking analysis. The resulting compounds 1-3 and a phenolic-enriched fraction of the extract, 2.9-fold more active than the starting material, may be suitable as non-toxic and inexpensive alternatives for the control of deleterious enzymatic darkening.

Cytotoxic Activity of Extracts from Plants of Central Argentina on Sensitive and Multidrug-Resistant Leukemia Cells: Isolation of an Active Principle from Gaillardia megapotamica.

Plants are a significant reservoir of cytotoxic agents, including compounds with the ability to interfere with multidrug-resistant (MDR) cells. With the aim of finding promising candidates for chemotherapy, 91 native and naturalized plants collected from the central region of Argentina were screened for their cytotoxic effect toward sensitive and MDR P-glycoprotein (P-gp) overexpressing human leukemia cells by means of MTT assays. The ethanol extracts obtained from Aldama tucumanensis, Ambrosia elatior, Baccharis artemisioides, Baccharis coridifolia, Dimerostemma aspilioides, Gaillardia megapotamica, and Vernonanthura nudiflora presented outstanding antiproliferative activity at 50 µg/mL, with inhibitory values from 93 to 100%, when tested on the acute lymphoblastic leukemia (ALL) cell line CCRF-CEM and the resistant derivative CEM-ADR5000, while 70-90% inhibition was observed against the chronic myelogenous leukemia (CML) cell K562 and its corresponding resistant subline, Lucena 1. Subsequent investigation showed these extracts to possess marked cytotoxicity with IC50 values ranging from 0.37 to 29.44 µg/mL, with most of them being below 7 µg/mL and with ALL cells, including the drug-resistant phenotype, being the most affected. G. megapotamica extract found to be one of the most effective and bioguided fractionation yielded helenalin (1). The sesquiterpene lactone displayed IC50 values of 0.63, 0.19, 0.74, and 0.16 µg/mL against K562, CCRF-CEM, Lucena 1, and CEM/ADR5000, respectively. These results support the potential of these extracts as a source of compounds for treating sensitive and multidrug-resistant leukemia cells and support compound 1 as a lead for developing effective anticancer agents.

P53 tumor suppressor is required for efficient execution of the death program following treatment with a cytotoxic limonoid obtained from Melia azedarach.

This work examines the antitumor activity of an isomeric mixture (1), composed of the limonoids meliartenin and its interchangeable isomer 12-hydroxyamoorastatin. The results obtained showed that 1 displayed outstanding cytotoxic activity against CCRF-CEM, K562, A549 and HCT116 cells, with a highly selective effect on the latter, with an IC50 value of 0.2 µM. Based on this finding, HCT116 cells were selected to study the mechanism of action of 1. Cell cycle analysis revealed that 1 induced sustained arrest in the S-phase, which was followed by the triggering of apoptotic cell death and reduced clonogenic capacity. This cytotoxicity was seen to be preceded by the upregulation of the tumor suppressor p53 and its target effector p21. In addition, it was found that p53 expression was required for efficient cell death induction, and thus that the toxicity of 1 relies mainly on p53-dependent mechanisms. Taken together, these findings position 1 as a potent antitumor agent, with potential for the development of novel chemotherapeutic drugs based on the induction of S-phase arrest.

IFNß produced by TLR4-activated tumor cells is involved in improving the antitumoral immune response.

Toll-like receptor (TLR) ligands may be a valuable tool to promote antitumor responses by reinforcing antitumor immunity. In addition to their expression in immune cells, functional TLRs are also expressed by many cancer cells, but their significance has been controversial. In this study, we examined the action of TLR ligands on tumor pathophysiology as a result of direct tumor cell effects. B16 murine melanoma cells were stimulated in vitro with a TLR4 ligand (LPS-B16) prior to inoculation into TLR4-deficient mice (Tlr4 (lps-del)). Under such conditions, B16 cells yielded smaller tumors than nonstimulated B16 cells. The apoptosis/proliferation balance of the cells was not modified by TLR ligand treatment, nor was this effect compromised in immunocompromised nude mice. Mechanistic investigations revealed that IFNß was the critical factor produced by TLR4-activated tumor cells in mediating their in vivo outgrowth. Transcriptional analysis showed that TLR4 activation on B16 cells induced changes in the expression of type I IFN and type I IFN-related genes. Most importantly, culture supernatants from LPS-B16 cells improved the maturation of bone marrow-derived dendritic cells (BMDC) from TLR4-deficient mice, upregulating the expression of interleukin-12 and costimulatory molecules on those cells. BMDC maturation was blunted by addition of an IFNß-neutralizing antibody. Moreover, tumor growth inhibition observed in LPS-B16 tumors was abrogated in IFNAR1-deficient mice lacking a functional type I IFN receptor for binding IFN. Together, our findings show that tumor cells can be induced through the TLR4 pathway to produce IFN and positively contribute to the antitumoral immune response.