Patrícia Sampaio Tavares Veras

Different Leishmania species determine distinct profiles of immune and histopathological responses in CBA mice.

Most experimental studies on leishmaniasis compare two different inbred strains of mice that are resistant or susceptible to one species of Leishmania. In the present study we characterized some cytokines and nitric oxide production as well as histological changes related to resistance and susceptibility in isogenic CBA mice infected with Leishmania major or Leishmania amazonensis. CBA mice are capable of controlling infection with L. major, but they succumb to infection with L. amazonensis. Cells from susceptible L. amazonensis-infected CBA mice produced interleukin (IL)-4 and IL-10 but no interferon (IFN)-γ. On the other hand, resistant L. major-infected CBA mice produced IFN-γ and IL-10, but IL-4 was detected only in the first week of infection. Histopathological studies showed patterns of tissue responses at the site of the infection and in the draining lymph nodes that correlated with resistance or susceptibility. Resistant mice showed a mixed inflammatory cell infiltration and granulomas in the lesions, whereas in susceptible mice only heavily parasitized macrophages were seen. Our results indicate an important role of the parasite species in determining the pattern of immune response. L. amazonensis induces a Th2-type immune response, whereas L. major induces a Th1-type response. These factors must be identified and taken into account in the strategies for the development of vaccines against leishmaniasis. The model presented here will be useful for the study of such factors.

Differential properties of CBA/J mononuclear phagocytes recovered from an inflammatory site and probed with two different species of Leishmania

While CBA/J mice fail to be permissive to Leishmania amazonensis-driven pathogenic processes, they heal easily following Leishmania major infection. The early-phase events are crucial to the outcome of Leishmania infection and it is known that macrophages (Mphi) are important in infection control. In the present study we investigated the role of Mphi in driving CBA/J susceptibility to L. amazonensis. We performed kinetic studies and compared the capacity of L. amazonensis and L. major to infect Mphi. There was no difference in percentages of infection or parasite burden for 6 h between the two groups. In contrast, after 12 h we observed that infection was about twice as high in L. amazonensis- than in L. major-infected Mphi. In addition, rIFN-gamma added to the cultures induced nitric oxide (NO) production, and did not modify L. amazonensis infection, although the percentage of L. major infection was significantly reduced. This reduction in L. major infection is a TNF-alpha dependent mechanism as L. major-infected Mphi expressed twice as much TNF-alpha mRNA as L. amazonensis-infected cells, and anti-TNF-alpha reversed the IFN-gamma effect. Moreover, rTNF-alpha plus IFN-gamma were able to significantly reduce the percentage of L. amazonensis-infected cells but not to the same extent as in L. major infection. Despite having higher NO production than IFN-gamma-treated cells, AMG addition to IFN-gamma-plus TNF-alpha-treated cells only partially reversed the inhibition in L. major, but not in L. amazonensis infection. Thus, in this study, we demonstrated that L. amazonensis both inactivated and resisted innate and IFN-gamma-induced Mphi killing mechanisms, indicating that the nature of the parasite and its interaction with Mphi could determine immune response polarization.

Comparison of two commercial vaccines against visceral leishmaniasis in dogs from endemic areas: IgG, and subclasses, parasitism, and parasite transmission by xenodiagnosis

BACKGROUND The incidence of zoonotic canine visceral leishmaniasis (CVL) would decrease if dogs were effectively vaccinated; however, additional data on the efficacy of canine vaccines are required for their approved preventative use. PURPOSE To prospectively evaluate vaccination outcomes using two products commercially available in Brazil, with respect to adverse reactions (reactogenicity), humoral response, disease signs, parasitism, and parasite infectiousness in naturally exposed pet dogs in an endemic area of visceral leishmaniasis (VL). METHODS From 2010 to 2012, healthy dogs were vaccinated with Leishmune(®) (50 animals) or Leish-Tec(®) (50 animals). Each dog was examined to identify clinical signs during peri- and post-vaccination procedures every 2 months for 11 months to identify the presence of parasites or parasite DNA in splenic samples using culturing or PCR, respectively. Levels of anti-Leishmania IgG, IgG1, and IgG2 were quantified in sera by ELISA and infectiousness was assessed by xenodiagnosis. RESULTS Adverse effects occurred in 2.2% (1/45) and 13.0% (6/46) of the animals in the Leishmune(®) and Leish-Tec(®) groups, respectively. IgG levels peaked on the 21st day following the first dose of Leishmune(®) and on the 21st day after the second dose of Leish-Tec(®). The final seropositivity rate for IgG was 32.5% (13/40) and 30.9% (13/42) in the Leishmune(®) and Leish-Tec(®) groups, respectively. The Leishmune(®) group presented higher levels of IgG1 and IgG2 compared to the Leish-Tec(®) group (p<0.001), and ELISA reactivity in both vaccinated groups was significantly lower (p<0.001) than in infected positive control dogs. Parasitism was observed in 12.2% (5/41) of the Leishmune(®) group, and 7.9% (3/38) of the Leish-Tec(®) group, with xenodiagnostic transmission rates of Leishmania to Lutzomyia longipalpis of 5.1% (2/39), and 5.4% (2/37), respectively. CONCLUSIONS No significant differences were observed in dogs vaccinated with Leishmune(®) or Leish-Tec(®), with respect to LVC clinical aspects, parasitism, IgG seropositivity, or dog infectiousness. The Leishmune(®)-vaccinated animals presented higher levels of IgG, IgG1, and IgG2. The animals vaccinated with Leish-Tec(®) exhibited adverse reactions with greater frequency and severity.

Advances in Development of New Treatment for Leishmaniasis.

Leishmaniasis is a neglected infectious disease caused by several different species of protozoan parasites of the genus Leishmania. Current strategies to control this disease are mainly based on chemotherapy. Despite being available for the last 70 years, leishmanial chemotherapy has lack of efficiency, since its route of administration is difficult and it can cause serious side effects, which results in the emergence of resistant cases. The medical-scientific community is facing difficulties to overcome these problems with new suitable and efficient drugs, as well as the identification of new drug targets. The availability of the complete genome sequence of Leishmania has given the scientific community the possibility of large-scale analysis, which may lead to better understanding of parasite biology and consequent identification of novel drug targets. In this review we focus on how high-throughput analysis is helping us and other groups to identify novel targets for chemotherapeutic interventions. We further discuss recent data produced by our group regarding the use of the high-throughput techniques and how this helped us to identify and assess the potential of new identified targets.

Cohabitation of Leishmania amazonensis and Coxiella burnetii

Intracellular pathogens customize the composition and function of the vacuoles they occupy, and can arrest or distort vacuolar maturation. In doubly infected cells, vacuoles that contain two different parasites can be used to test for exclusionary mechanisms, for expression of vacuolar phenotypes that permit or restrict fusion, and for the survival of pathogens targeted to an unusual cellular compartment.

Qualitative and quantitative polymerase chain reaction (PCR) for detection of Leishmania in spleen samples from naturally infected dogs

Because infected dogs are widely considered to be the main domestic reservoir for Leishmania infantum (syn Leishmania chagasi) parasites in Brazil, the diagnosis of canine visceral leishmaniasis (CVL) must be made both accurately and promptly. The present study attempted to standardize a conventional polymerase chain reaction (cPCR) protocol for the detection of L. infantum DNA in canine spleen samples. Quantitative PCR (qPCR) technique was used to confirm the presence of Leishmania DNA in the canine spleen fragments. A comparison was made between the efficacies of these molecular diagnostic techniques and conventional parasitological and serological methods. cPCR protocols for spleen samples were standardized using primers that amplify a 145 bp fragment, located at the parasite kinetoplast minicircle. The genus specificity of the cPCR protocol was assessed by its inability to amplify the DNA of other common canine pathogens, such as Ehrlichia canis, Babesia canis, Toxoplasma gondii and Trypanosoma cruzi. cPCR protocol sensitivity was tested by assessing the reaction detection limit, determined to be 10 fg of L. infantum reference strain DNA, which corresponds to a range of 0.03-0.1 parasites per fragment. Standardized cPCR protocol was used to detect the presence of Leishmania in 45 dog spleen samples. Our results showed that 40% of the spleen fragment cultures were positive for Leishmania parasites, 58% of the dog serum samples tested positive using ELISA, and parasite DNA was detected in 44% using qPCR, while 47% of the spleen samples using cPCR. Diagnostic methods performance was assessed and revealed a better degree of ascertainment for cPCR when compared to other diagnostic methods. The sensitivity of ELISA was 83.3%, qPCR was 83.3%, and cPCR was 88.9%; PPV for ELISA was 57.7%, qPCR was 75% and cPCR was 76.2%; the Kappa coefficients were found to be 0.40 (fair) for ELISA, 0.64 (substantial) for qPCR and 0.68 (substantial) for cPCR. In both oligosymptomatic and polysymptomatic dogs, cPCR revealed the better performance analysis when compared to other diagnostic methods. The findings presented herein establish cPCR as the most indicated test to detect Leishmania when compared to the other two diagnostic methods evaluated. Despite the fact that the qPCR protocol provides a highly accurate quantification of parasites when targeting the SSU rRNA gene, this technique does not significantly improve the diagnosis of CVL when compared with the performance of the cPCR protocol, which focused on the kinetoplast minicircle.

Evaluating the accuracy of molecular diagnostic testing for Canine Visceral Leishmaniasis using latent class analysis

Host tissues affected by Leishmania infantum have differing degrees of parasitism. Previously, the use of different biological tissues to detect L. infantum DNA in dogs has provided variable results. The present study was conducted to evaluate the accuracy of molecular diagnostic testing (qPCR) in dogs from an endemic area for canine visceral leishmaniasis (CVL) by determining which tissue type provided the highest rate of parasite DNA detection. Fifty-one symptomatic dogs were tested for CVL using serological, parasitological and molecular methods. Latent class analysis (LCA) was performed for accuracy evaluation of these methods. qPCR detected parasite DNA in 100% of these animals from at least one of the following tissues: splenic and bone marrow aspirates, lymph node and skin fragments, blood and conjunctival swabs. Using latent variable as gold standard, the qPCR achieved a sensitivity of 95.8% (CI 90.4–100) in splenic aspirate; 79.2% (CI 68–90.3) in lymph nodes; 77.3% (CI 64.5–90.1) in skin; 75% (CI 63.1–86.9) in blood; 50% (CI 30–70) in bone marrow; 37.5% (CI 24.2–50.8) in left-eye; and 29.2% (CI 16.7–41.6) in right-eye conjunctival swabs. The accuracy of qPCR using splenic aspirates was further evaluated in a random larger sample (n = 800), collected from dogs during a prevalence study. The specificity achieved by qPCR was 76.7% (CI 73.7–79.6) for splenic aspirates obtained from the greater sample. The sensitivity accomplished by this technique was 95% (CI 93.5–96.5) that was higher than those obtained for the other diagnostic tests and was similar to that observed in the smaller sampling study. This confirms that the splenic aspirate is the most effective type of tissue for detecting L. infantum infection. Additionally, we demonstrated that LCA could be used to generate a suitable gold standard for comparative CVL testing.

Low CXCL13 Expression, Splenic Lymphoid Tissue Atrophy and Germinal Center Disruption in Severe Canine Visceral Leishmaniasis

Visceral leishmaniasis is associated with atrophy and histological disorganization of splenic compartments. In this paper, we compared organized and disorganized splenic lymphoid tissue from dogs naturally infected with Leishmania infantum assessing the size of the white pulp compartments, the distribution of T, B and S100+ dendritic cells, using immunohistochemistry and morphometry and the expression of CCR7 and the cytokines, CXCL13, lymphotoxin (LT)-α, LT-β, CCL19, CCL21, TNF-α, IL-10, IFN-γ and TGF-β, using by real time RT-PCR. The lymphoid follicles and marginal zones were smaller (3.2 and 1.9 times, respectively; Mann-Whitney, P<0.02) in animals with disorganized splenic tissue in comparison to those with organized splenic lymphoid tissue. In spleens with disorganized lymphoid tissue, the numbers of T cells and S100+ dendritic cells were decreased in the follicles, and the numbers of B cells were reduced in both the follicles and marginal zones. CXCL13 mRNA expression was lower in animals with disorganized lymphoid tissue (0.5±0.4) compared to those with organized lymphoid tissue (2.7±2.9, both relative to 18S expression, P = 0.01). These changes in the spleen were associated with higher frequency of severe disease (7/12) in the animals with disorganized than in animals with organized (2/13, Chi-square, P = 0.01) splenic lymphoid tissue. The data presented herein suggest that natural infection with Leishmania infantum is associated with the impairment of follicular dendritic cells, CXCL13 expression, B cell migration and germinal center formation and associates these changes with severe clinical forms of visceral leishmaniasis. Furthermore the fact that this work uses dogs naturally infected with Leishmania infantum emphasizes the relevance of the data presented herein for the knowledge on the canine and human visceral leishmaniasis.

Cell-to-cell transfer of Leishmania amazonensis amastigotes is mediated by immunomodulatory LAMP-rich parasitophorous extrusions

The last step of Leishmania intracellular life cycle is the egress of amastigotes from the host cell and their uptake by adjacent cells. Using multidimensional live imaging of long‐term‐infected macrophage cultures we observed that Leishmania amazonensis amastigotes were transferred from cell to cell when the donor host macrophage delivers warning signs of imminent apoptosis. They were extruded from the macrophage within zeiotic structures (membrane blebs, an apoptotic feature) rich in phagolysosomal membrane components. The extrusions containing amastigotes were selectively internalized by vicinal macrophages and the rescued amastigotes remain viable in recipient macrophages. Host cell apoptosis induced by micro‐irradiation of infected macrophage nuclei promoted amastigotes extrusion, which were rescued by non‐irradiated vicinal macrophages. Using amastigotes isolated from LAMP1/LAMP2 knockout fibroblasts, we observed that the presence of these lysosomal components on amastigotes increases interleukin 10 production. Enclosed within host cell membranes, amastigotes can be transferred from cell to cell without full exposure to the extracellular milieu, what represents an important strategy developed by the parasite to evade host immune system.

Virulent Mycobacterium fortuitum Restricts NO Production by a Gamma Interferon-Activated J774 Cell Line and Phagosome-Lysosome Fusion

ABSTRACT The virulence of different isolates of Mycobacterium has been associated with two morphologically distinguishable colonial variants: opaque (SmOp) and transparent (SmTr). In this report we used an in vitro assay to compare macrophage (Mφ) responses to SmOp and SmTr Mycobacterium fortuitum variants, taking advantage of the fact that these variants were derived from the same isolate. Cells preactivated or not with gamma interferon (IFN-γ) were infected with SmOp or SmTr M. fortuitum. We showed that SmOp and SmTr induced different levels of nitric oxide (NO) production by IFN-γ-stimulated Mφ. Indeed, the amount of IFN-γ-induced NO production by J774 cells was 4.8 to 9.0 times higher by SmOp (23.1 to 37.7 μM) compared to SmTr infection (3.9 to 4.8 μM) (P = 0.0332), indicating that virulent SmTr bacilli restricted NO production. In addition, IFN-γ-induced NO production by Mφ was higher when correlated with reduction of only avirulent SmOp bacillus viability. SNAP (S-nitroso-N-acetyl-dl-penicillamine)-induced NO production did not modify SmTr viability, indicating its resistance to nitrogen radicals. Electron microscopy studies were performed to evaluate the capacity of phagosomes to fuse with lysosomes labeled with bovine serum albumin-colloidal gold particles. By 24 h postinfection, 69% more phagosome-containing SmOp variant had fused with lysosomes compared to the SmTr-induced phagosomes. In conclusion, these data indicate that virulent SmTr bacilli may escape host defense by restricting IFN-γ-induced NO production, resisting nitrogen toxic radicals, and limiting phagosome fusion with lysosomes.