Selective whole-genome amplification reveals population genetics of Leishmania braziliensis directly from patient skin biopsies.

In Brazil, Leishmania braziliensis is the main causative agent of the neglected tropical disease, cutaneous leishmaniasis (CL). CL presents on a spectrum of disease severity with a high rate of treatment failure. Yet the parasite factors that contribute to disease presentation and treatment outcome are not well understood, in part because successfully isolating and culturing parasites from patient lesions remains a major technical challenge. Here we describe the development of selective whole genome amplification (SWGA) for Leishmania and show that this method enables culture-independent analysis of parasite genomes obtained directly from primary patient skin samples, allowing us to circumvent artifacts associated with adaptation to culture. We show that SWGA can be applied to multiple Leishmania species residing in different host species, suggesting that this method is broadly useful in both experimental infection models and clinical studies. SWGA carried out directly on skin biopsies collected from patients in Corte de Pedra, Bahia, Brazil, showed extensive genomic diversity. Finally, as a proof-of-concept, we demonstrated that SWGA data can be integrated with published whole genome data from cultured parasite isolates to identify variants unique to specific geographic regions in Brazil where treatment failure rates are known to be high. SWGA provides a relatively simple method to generate Leishmania genomes directly from patient samples, unlocking the potential to link parasite genetics with host clinical phenotypes.

Inhibition of gamma-secretase activity without interfering in Notch signalling decreases inflammatory response in patients with cutaneous leishmaniasis.

Cutaneous leishmaniasis (CL) patients present an exacerbated inflammatory response associated with tissue damage and ulcer development. Increasing numbers of patients have exhibited treatment failure, which remains not well understood. We hypothesized that adjuvant anti-inflammatory therapy would benefit CL patients. The aim of the present study was to investigate the contribution of Notch signalling and gamma-secretase activity to the inflammatory response observed in CL patients. Notch signalling is a molecular signalling pathway conserved among animal species. Gamma-secretase forms a complex of proteins that, among other pathways, modulates Notch signalling and immune response. We found that Notch 1 cell receptor signalling protects against the pathologic inflammatory response, and JLK6, a gamma-secretase inhibitor that does not interfere with Notch signalling, was shown to decrease the in-vitro inflammatory response in CL. Our data suggest that JLK6 may serve as an adjuvant treatment for CL patients.

The Role of NK Cells in the Control of Viral Infection in HTLV-1 Carriers.

The cytotoxic activities of CD8+ T cells have been considered the main defense mechanism against the human T lymphotropic virus type 1 (HTLV-1). As with CD8+ T cells, NK cells can perform cytotoxic degranulation with production of cytotoxic mediators, such as perforins and granzymes. NK cells are also responsible for antibody-dependent cellular cytotoxicity (ADCC) against infected cells, but few studies have evaluated the role of NK cells in HTLV-1 infection. The aim of this study was to characterize the subsets and measure the frequency of NK cells in HTLV-1 carriers (HC) and in patients with HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) and correlate these findings with the proviral load and development of HAM/TSP. The diagnosis of HTLV-1 infection was performed with a detection antibody against viral antigens by ELISA and confirmed by Western blot. Phenotypic characterization of NK cells was performed by flow cytometry. The frequencies of CD56+, CD56+CD3-, CD56+CD16+, and CD56dim cells were decreased in HAM/TSP patients. The frequency of CD56+CD3- cells was inversely correlated with proviral load in HC but not in HAM/TSP patients. HAM/TSP patients showed decreased frequency of CD56+ and CD56dim cells expressing CD16, the main receptor for ADCC. These data indicate that NK cells may play a key role in the control of HTLV-1 infection by preventing the progression of HC to HAM/TSP.

Inhibitory activity of pentacyano(isoniazid)ferrate(II), IQG-607, against promastigotes and amastigotes forms of Leishmania braziliensis.

M. tuberculosis and parasites of the genus Leishmania present the type II fatty acid biosynthesis system (FASII). The pentacyano(isoniazid)ferrate(II) compound, named IQG-607, inhibits the enzyme 2-trans-enoyl-ACP(CoA) reductase from M. tuberculosis, a key component in the FASII system. Here, we aimed to evaluate the inhibitory activity of IQG-607 against promastigote and amastigote forms of Leishmania (Viannia) braziliensis isolated from patients with different clinical forms of L. braziliensis infection, including cutaneous, mucosal and disseminated leishmaniasis. Importantly, IQG-607 inhibited the proliferation of three different isolates of L. braziliensis promastigotes associated with cutaneous, mucosal and disseminated leishmaniasis. The IC50 values for IQG-607 ranged from 32 to 75 µM, for these forms. Additionally, IQG-607 treatment decreased the proliferation of intracellular amastigotes in infected macrophages, after an analysis of the percentage of infected cells and the number of intracellular parasites/100 cells. IQG-607 reduced from 58% to 98% the proliferation of L. braziliensis from cutaneous, mucosal and disseminated strains. Moreover, IQG-607 was also evaluated regarding its potential toxic profile, by using different cell lines. Cell viability of the lineages Vero, HaCat and HepG2 was significantly reduced after incubation with concentrations of IQG-607 higher than 2 mM. Importantly, IQG-607, in a concentration of 1 mM, did not induce DNA damage in HepG2 cells, when compared to the untreated control group. Future studies will confirm the mechanism of action of IQG-607 against L. braziliensis.

Age modifies the immunologic response and clinical presentation of American tegumentary leishmaniasis.

Leishmania (Viannia) braziliensis is the main causal agent of American tegumentary leishmaniasis (ATL) that may present as cutaneous, mucosal, or disseminated cutaneous leishmaniasis. The disease is highly prevalent in young males and there is a lack of studies of ATL in the elderly. Herein, we compared clinical manifestations, immunologic response, and response to antimony therapy between patients > 60 years of age (N = 58) and patients who were 21-30 years of age (N = 187). The study was performed in Corte de Pedra, Bahia, Brazil, a well-known area of L. braziliensis transmission. Cytokine production by cultured peripheral blood mononuclear cells stimulated with soluble Leishmania antigen was performed. Elderly subjects more frequently had a previous history of cutaneous leishmaniasis, large lesions, or mucosal leishmaniasis, and they were less likely to have lymphadenopathy. There was no difference regarding gender and response to therapy. Peripheral blood mononuclear cells from elderly subjects produced a similar amount of tumor necrosis factor than young patients but they produced less interferon-gamma and more interleukin-10 than young subjects. We concluded that elderly patients with cutaneous leishmaniasis should be searched for mucosal or disseminated leishmaniasis. The decreased interferon-gamma production and increase in interleukin-10 observed in elderly patients may contribute to parasite persistence and L. braziliensis infection dissemination.

Functional activity of monocytes and macrophages in HTLV-1 infected subjects.

The Human T lymphotropic virus type-1 (HTLV-1) infects predominantly T cells, inducing proliferation and lymphocyte activation. Additionally, HTLV-1 infected subjects are more susceptible to other infections caused by other intracellular agents. Monocytes/macrophages are important cells in the defense against intracellular pathogens. Our aims were to determine the frequency of monocytes subsets, expression of co-stimulatory molecules in these cells and to evaluate microbicidal ability and cytokine and chemokine production by macrophages from HTLV-1 infected subjects. Participants were 23 HTLV-1 carriers (HC), 22 HAM/TSP patients and 22 healthy subjects (HS) not infected with HTLV-1. The frequencies of monocyte subsets and expression of co-stimulatory molecules were determined by flow cytometry. Macrophages were infected with L. braziliensis or stimulated with LPS. Microbicidal activity of macrophages was determined by optic microscopy. Cytokines/chemokines from macrophage supernatants were measured by ELISA. HAM/TSP patients showed an increase frequency of intermediate monocytes, but expression of co-stimulatory molecules was similar between the groups. Macrophages from HTLV-1 infected individuals were infected with L. braziliensis at the same ratio than macrophages from HS, and all the groups had the same ability to kill Leishmania parasites. However, macrophages from HTLV-1 infected subjects produced more CXCL9 and CCL5, and less IL-10 than cells from HS. While there was no correlation between IFN-γ and cytokine/chemokine production by macrophages, there was a correlation between proviral load and TNF and CXCL10. These data showed a dissociation between the inflammatory response and microbicidal ability of macrophages from HTLV-1 infected subjects. While macrophages ability to kill an intracellular pathogen did not differ among HTLV-1 infected subjects, these cells secreted high amount of chemokines even in unstimulated cultures. Moreover the increasing inflammatory activity of macrophages was similar in HAM/TSP patients and HC and it was related to HTLV-1 proviral load rather than the high IFN-γ production observed in these subjects.