Potent stimulation of the innate immune system by a Leishmania brasiliensis recombinant protein.

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 a Leishmania brasiliensis gene 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.

Assessment of the serodiagnostic potential of nine novel proteins from Mycobacterium tuberculosis.

To identify antigens that would improve the accuracy of serological diagnosis of active tuberculosis, we cloned the genes encoding nine potentially immunogenic secreted or surface-associated proteins of Mycobacterium tuberculosis. Recombinant proteins were reacted with sera from HIV-negative individuals with extrapulmonary tuberculosis (EP-TB) or HIV-positive individuals with pulmonary tuberculosis (TBH). Specific and high level antibody responses were obtained for four recombinant proteins, of which antigen GST-822 was recognized by 60% of EP-TB and 42% of TBH and antigen MBP-506 was recognized by 45% of EP-TB and 61% of TBH. These results suggest that these proteins are strong candidates as subunits in a polyvalent serodiagnostic test.

Multiepitope synthetic peptide and recombinant protein for the detection of antibodies to Trypanosoma cruzi in patients with treated or untreated Chagas' disease.

A tetrapeptide and a recombinant protein, each representing 4 immunodominant epitopes of Trypanosoma cruzi, were tested by use of ELISA for the detection of serum antibodies. Sera from individuals with Chagas' disease, including persons untreated and successfully or unsuccessfully treated, were tested. These assays detected antibody in 100% of the parasitemias. The antibody reactivity decreased based on the success of treatment. Higher sensitivity was observed for tetrapeptide/recombinant protein assays than for lysate-based ELISA, and specificity was improved, particularly with Leishmania sera. The results indicate that multiepitope antigens provide a more sensitive and specific alternative to lysate for detection of anti-T. cruzi antibodies, as required for developing blood screening assays.

Association of Leishmania heat shock protein 83 antigen and immunoglobulin G4 antibody titers in Brazilian patients with diffuse cutaneous leishmaniasis.

Diffuse cutaneous leishmaniasis (DCL) is characterized by the presence of numerous nonulcerated nodules and plaques containing large numbers of Leishmania amazonensis parasites and few lymphoid elements. The immune responses of DCL patients reflect severe antigen-specific T-cell deficiencies, while the antibody response to Leishmania antigens is often accentuated. We report herein on the Leishmania antigen-specific antibody subclass distribution in DCL patients and demonstrate that a dominant antigen contributing to the biased immunoglobulin G4 antibody subclass in sera of DCL patients is Leishmania heat shock protein 83.

Cloning and expression of a Leishmania donovani gene instructed by a peptide isolated from major histocompatibility complex class II molecules of infected macrophages.

The studies reported here describe the isolation of peptides from MHC class II molecules of murine macrophages infected with Leishmania donovani, and the use of the derived peptide sequences to rescue the pathogen peptide donor protein. The isolation of the peptides was carried out by comparing the RP HPLC profile of peptides extracted from infected macrophages with the peptides extracted from noninfected cells. Several distinct HPLC peaks unique to infected macrophages were sequenced. One of the peptides that was not homologous to any known protein was used to instruct the designing of an oligonucleotide sense primer that was used in combination with an oligo dT nucleotide (anti-sense primer) to amplify by PCR a DNA fragment from L. donovani cDNA. The amplified DNA fragment was cloned and used as a probe to screen a L. donovani cDNA library. The cloned gene (Ld peptide gene) has an open reading frame of 525 bp and has no homology with any known protein/gene sequence. Northern blot analyses indicated that the Ld peptide/gene is broadly distributed and expressed among species of the Leishmania genus, in both the amastigote and promastigote life cycle forms. Using the pGEX 2T vector, the gene was expressed and the relationship of the purified recombinant protein with L. donovani was confirmed using both antibody and T cell responses from immunized or infected animals. The gene encodes a 23-kD molecule (Ldp 23) associated with the cell surface of L. donovani promastigotes. In addition, T cells purified from the lymph nodes of BALB/c mice immunized with L. donovani or infected with L. major, and from CBA/J mice infected with L. amazonensis were stimulated to proliferate by the recombinant Ldp 23 and produced high levels of IFN-gamma and no IL 4. This observation suggests that the Ldp 23 is an interesting parasite molecule for the studies concerning the host/parasite interaction because the Th1 pattern of cytokine response that it induces is correlated with resistance to Leishmania infections. These results clearly point to an alternative strategy for the purification of proteins useful for the development of both vaccines and immunological diagnostic tools not only against leishmaniasis but also for other diseases caused by intracellular pathogens.

Transforming growth factor-beta in leishmanial infection: a parasite escape mechanism.

The course of infection with the protozoan parasite Leishmania is determined in part by their early replication in macrophages, the exclusive host cells for these organisms. Although factors contributing to the survival of Leishmania are not well understood, cytokines influence the course of infection. Transforming growth factor-beta (TGF-beta) is a multipotential cytokine with diverse effects on cells of the immune system, including down-regulation of certain macrophage functions. Leishmanial infection induced the production of active TGF-beta, both in vitro and in vivo. TGF-beta was important for determining in vivo susceptibility to experimental leishmanial infection.