Biological Activities of Extracts from Ageratum fastigiatum: Phytochemical Study and In Silico Target Fishing Approach.

In the present study, the ethanolic extract from aerial parts of Ageratum fastigiatum was evaluated in vitro against epimastigote forms of Trypanosoma cruzi (Y strain), promastigote forms of Leishmania amazonensis (PH8 strain), and L. chagasi (BH400 strain). The extract was also evaluated against Staphylococcus aureus (ATCC 25 923), Escherichia coli (ATCC 11 775), Pseudomonas aeruginosa (ATCC 10 145), and Candida albicans (ATCC 36 802). The phytochemical screening was performed by thin-layer chromatography and high-performance liquid chromatography. The extract was fractionated using flash preparative chromatography. The ethanolic extract showed activity against T. cruzi, L. chagasi, and L. amazonensis and antimicrobial activity against S. aureus, E. coli, P. aeruginosa, and C. albicans. The phytochemical screening revealed coumarins, terpenes/sterols, and flavonoids in the ethanolic extract. In addition, the coumarin identified as ayapin was isolated from this extract. We also performed in silico prediction of potential biological activities and targets for compounds previously found in A. fastigiatum. Several predictions were confirmed both retrospectively and prospectively by experimental results described here or elsewhere. Some activities described in the in silico target fishing approach were validated by the ethnopharmacological use and known biological properties. Some new activities and/or targets were predicted and could guide future studies. These results suggest that A. fastigiatum can be an interesting source of substances with antiparasitic and antimicrobial activities.

Lactococcus lactis FNBPA+ (pValac:e6ag85a) Induces Cellular and Humoral Immune Responses After Oral Immunization of Mice.

The development of a new vaccine strategy against tuberculosis is urgently needed and has been greatly encouraged by the scientific community worldwide. In this work, we constructed a lactococcal DNA vaccine based on the fusion of two Mycobacterium tuberculosis antigens, ESAT-6 and Ag85A, and examined its immunogenicity. The coding sequences of the ESAT-6 and Ag85A genes were fused and cloned into the eukaryotic expression pValac vector, and the functionality of the vector was confirmed in vitro. Then, L. lactis FnBPA+ (pValac:e6ag85a) was obtained and used for oral immunization of mice. This strain induced significant increases in IFN-γ, TNF-α, and IL-17 cytokines in stimulated splenocyte cultures, and significant production of antigen-specific sIgA was observed in the colonic tissues of immunized mice. We demonstrated that L. lactis FnBPA+ (pValac:e6ag85a) generated a cellular and humoral immune response after oral immunization of mice. The strategy developed in this work may represent an interesting DNA mucosal vaccine candidate against tuberculosis, using the fusion of two highly immunogenic antigens delivered by safe lactic acid bacteria.

Reverse and structural vaccinology approach to design a highly immunogenic multi-epitope subunit vaccine against Streptococcus pneumoniae infection.

Streptococcus pneumoniae is a pathogen that resides in the upper respiratory tract of healthy individuals, maintaining a commensal relationship with its host. However, the virulent form may be the etiology of pneumonia, meningitis, bacteremia, and other respiratory tract infections. Streptococcal diseases are preventable by vaccination; but currently available vaccines have some drawbacks, especially due to the high capsule variability of streptococci strains. Thus, an effective prevention strategy continues to be the focus of extensive research. In our work, several bioinformatics tools were used to identify immunogenic peptides from a selected pool of 46 conserved proteins from Streptococcus pneumoniae. In silico analysis showed that 10 proteins had epitopes with affinity for B and T lymphocytes, which were present in at least 26 different pathogens serotypes and were considered promiscuous. The multi-epitope protein, designated HC44, was designed based on these epitopes and specific linkers to improve stability and exposure to T lymphocytes. The recombinant HC44 protein was expressed in E.coli and Swiss-Webster mice were immunised by intraperitoneal injection. Immunisation with the multi-epitope HC44 protein resulted in the production of very high levels of IgG with title superior to 1/1.200.000. However, subtype IgG was highly unbalanced toward IgG1 and no protection was afforded after challenge with S.pneumoniae in a sepsis model. Thus, our strategy has been effective in constructing a highly antigenic protein but novel immunisation strategies should be investigated to reorient the immune system toward a protective response.

Development of a new DNA vaccine based on mycobacterial ESAT-6 antigen delivered by recombinant invasive Lactococcus lactis FnBPA+.

The use of the food-grade bacterium Lactococcus lactis as a vehicle for the oral delivery of DNA vaccine plasmids constitutes a promising strategy for vaccination. The delivery of DNA plasmids into eukaryotic cells is of critical importance for subsequent DNA expression and effectiveness of the vaccine. In this context, the use of the recombinant invasive L. lactis FnBPA+ (fibronectin-binding protein A) strain for the oral delivery of the eukaryotic expression vector vaccination using lactic acid bacteria (pValac), coding for the 6-kDa early secreted antigenic target (ESAT-6) gene of Mycobacterium tuberculosis, could represent a new DNA vaccine strategy against tuberculosis. To this end, the ESAT-6 sequence was cloned into the pValac vector; the L. lactis fibronectin-binding protein A (FnBPA)+ (pValac:ESAT-6) strain was obtained, and its immunological profile was checked in BALB/c mice. This strain was able to significantly increase interferon gamma (IFN-γ) production in spleen cells, showing a systemic T helper 1 (Th1) cell response. The mice also showed a significant increase in specific secretory immunoglobulin A (sIgA) production in colon tissue and fecal extracts. Thus, this is the first time that L. lactis has been used to deliver a plasmid DNA harboring a gene that encodes an antigen against tuberculosis through mucous membranes.

Trypanosoma cruzi-induced activation of functionally distinct αß and γδ CD4- CD8- T cells in individuals with polar forms of Chagas' disease.

CD4(-) CD8(-) (double-negative [DN]) T cells have recently been shown to display important immunological functions in human diseases. They express γδ or αß T-cell receptors that recognize lipid/glycolipid antigens presented via the nonclassical major histocompatibility complex molecules of the CD1 family. We recently demonstrated that while αß DN T cells serve primarily to express inflammatory cytokines, γδ DN T cells express mainly interleukin-10 (IL-10) in patients with cutaneous leishmaniasis. We also demonstrated a correlation between DN T cells and the expression of gamma interferon in the acute phase of Trypanosoma cruzi experimental infection. In this work, we sought to investigate whether αß or γδ DN T cells display distinct immunoregulatory potentials in patients with polar forms of human Chagas' disease. Our data showed that in vitro infection with T. cruzi leads to expansion of DN T cells in patients with the indeterminate and severe cardiac clinical forms of the disease. However, while αß DN T cells primarily produce inflammatory cytokines in both forms of the disease, γδ DN T cells display a marked, significant increase in antigen-specific IL-10 expression in indeterminate patients relative to cardiac patients. Finally, higher frequencies of the IL-10-producing γδ DN T cells were correlated with improved clinical measures of cardiac function in the patients, suggesting a protective role for these cells in Chagas' disease. Taken together, these data show distinct functional characteristics for αß and γδ DN T cells associated with distinct morbidity rates and clinical forms in human Chagas' disease.

Systemic leukocyte activation in patients with central giant cell lesions.

BACKGROUND: Central giant cell lesion (CGCL) is a reactive lesion of the jaws with an associated inflammatory infiltrate. Since cell circulation allows for intense communication between different compartments in the body, we investigated whether the CGCL would lead to phenotypic and/or functional changes in circulating leukocytes. METHODS: We obtained lymphocytes and monocytes from CGCL patients and control subjects, to evaluate cytokine and adhesion molecule expression using flow cytometry. RESULTS: Our results revealed that CD4(+) T cells and CD14(+) monocytes from CGCL express elevated levels of CD11a and CD11b, respectively, when compared with controls. The frequencies of CD4(+) T cells expressing interferon (IFN)-gamma and tumor necrosis factor (TNF)-alpha and the frequencies of CD4(+) and CD14(+) cells expressing interleukin (IL)-10 were increased in CGCL group, when compared with controls. CONCLUSIONS: Our data indicate that, although CGCL is a localized lesion, the patients show systemic functional alterations in circulating leukocytes, suggesting their role in the inflammatory pathogenesis of CGCL.