We have evaluated the ability of the Leishmania protein LeIF to influence the Th1/Th2 cytokine responses and the generation of LeIF-specific T cell clones in the absence of adjuvant. We characterized LeIF-specific T cell responses in Leishmania major-infected and uninfected BALB/c mice. These mice develop a strong Th2 response during infection with L. major. When lymph node cells from infected BALB/c mice were stimulated in vitro with LeIF, only IFN-γ (and no detectable IL-4) was found in the culture supernatant. In addition, LeIF down-regulated Leishmania Ag-specific IL-4 production by lymph node cells from infected BALB/c mice. Subsequently, Th responses were evaluated in naive BALB/c mice following immunization with LeIF. T cell clones derived from mice immunized with LeIF preferentially secreted IFN-γ. Finally, to understand the basis for the preferential Th1 cytokine bias observed with LeIF, the ability of LeIF to influence the early cytokine profile was evaluated in splenocytes of SCID mice. We found that LeIF stimulated fresh spleen cells from naive SCID mice to secrete IFN-γ by IL-12/IL-18-dependent mechanisms. The N-terminal half of the molecule (amino acid residues 1–226) maintained the ability to stimulate IFN-γ from splenocytes of SCID mice. Finally, we also demonstrated that LeIF was able to provide partial protection of BALB/c mice against L. major. Thus, our results suggest the potential of LeIF as a Th1-type adjuvant and as a therapeutic and prophylactic vaccine Ag for leishmaniasis when used with other leishmanial Ags.

Antibiotic resistance is at dangerous levels and increasing worldwide. The search for new antimicrobial drugs to counteract this problem is a priority for health institutions and organizations, both globally and in individual countries. Sarconesiopsis magellanica blowfly larval excretions and secretions (ES) are an important source for isolating antimicrobial peptides (AMPs). This study aims to identify and characterize a new S. magellanica AMP. RP-HPLC was used to fractionate ES, using C18 columns, and their antimicrobial activity was evaluated. The peptide sequence of the fraction collected at 43.7 min was determined by mass spectrometry (MS). Fluorescence and electronic microscopy were used to evaluate the mechanism of action. Toxicity was tested on HeLa cells and human erythrocytes; physicochemical properties were evaluated. The molecule in the ES was characterized as sarconesin II and it showed activity against Gram-negative (Escherichia coli MG1655, Pseudomonas aeruginosa ATCC 27853, P. aeruginosa PA14) and Gram-positive (Staphylococcus aureus ATCC 29213, Micrococcus luteus A270) bacteria. The lowest minimum inhibitory concentration obtained was 1.9 μM for M. luteus A270; the AMP had no toxicity in any cells tested here and its action in bacterial membrane and DNA was confirmed. Sarconesin II was documented as a conserved domain of the ATP synthase protein belonging to the Fli-1 superfamily. The data reported here indicated that peptides could be alternative therapeutic candidates for use in infections against Gram-negative and Gram-positive bacteria and eventually as a new resource of compounds for combating multidrug-resistant bacteria.

An improved whole cell pertussis vaccine, designated as Plow, which is low in endotoxicity due to a chemical extraction of lipo-oligosaccharide (LOS) from the outer membrane, was evaluated for safe...

The interaction of the innate immune system with the microbial world involves primarily two sets of molecules generally known as microbial pattern recognition receptors and microbial pattern recognition molecules, respectively. Examples of the former are the Toll receptors present particularly in macrophages and dendritic cells. Conversely, the microbial pattern recognition molecules are conserved protist homopolymers, such as bacterial lipopolysaccharides, lipoteichoic acids, peptidoglycans, glucans, mannans, unmethylated bacterial DNA, and double-strand viral RNA. However, for protists that lack most of these molecules, such as protozoans, the innate immune system must have evolved receptors that recognize other groups of microbial molecules. Here we present evidence that a highly purified protein encoded by aLeishmania brasiliensisgene may be one such molecule. This recombinant leishmanial molecule, a homologue of eukaryotic ribosomal elongation and initiation factor 4a (LeIF), strongly stimulates spleen cells from severe combined immunodeficient (SCID) mice to produce interleukin-12 (IL-12), IL-18, and high levels of gamma interferon. In addition, LeIF potentiates the cytotoxic activity of the NK cells of these animals. Because LeIF is a conserved molecule and because SCID mice lack T and B lymphocytes but have a normal innate immune system (normal reticuloendothelial system and NK cells), these results suggest that proteins may also be included as microbial pattern recognition molecules. The nature of the receptor involved in this innate recognition is unknown. However, it is possible to exclude the Toll receptor Tlr4 as a putative LeIF receptor because the gene encoding this receptor is defective in C3H/HeJ mice, the mouse strain used in the present studies.

Toll-like receptors (TLRs) are transmembrane proteins that are key regulators of innate and adaptive immune responses, particularly TLR4, and they have been identified as potential drug targets for the treatment of disease. Several low-molecular-weight compounds are being considered as new drug targets for various applications, including as immune modulators. Mygalin, a 417 Da synthetic bis-acylpolyamine, is an analog of spermidine that has microbicidal activity. In this study, we investigated the effect of mygalin on the innate immune response based on a virtual screening (VS) and molecular docking analysis. Bone marrow-derived macrophages and the cell lines J774A.1 and RAW 264.7 stimulated with lipopolysaccharide (LPS) were used to confirm the data obtained in silico. Virtual screening and molecular docking suggested that mygalin binds to TLR4 via the protein myeloid differentiation factor 2 (MD-2) and LPS. Macrophages stimulated by mygalin plus LPS showed suppressed gene expression of tumor necrosis factor (TNF-α), interleukine 6 (IL-6), cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS), as well as inhibition of signaling protein p65 of the nuclear factor κB (NF-κB), resulting in decreased production of nitric oxide (NO) and TNF-α. These results indicate that mygalin has anti-inflammatory potential, being an attractive option to be explored. In addition, we reinforce the importance of virtual screening analysis to assist in the discovery of new drugs.