Carolina Carvalho de Souza

Ecto-nucleotidase activities of promastigotes from leishmania (Viannia) braziliensis relates to parasite infectivity and disease clinical outcome.

Background Leishmania (Viannia) braziliensis has been associated with a broad range of clinical manifestations ranging from a simple cutaneous ulcer to destructive mucosal lesions. Factors leading to this diversity of clinical presentations are not clear, but parasite factors have lately been recognized as important in determining disease progression. Given the fact that the activity of ecto-nucleotidases correlates with parasitism and the development of infection, we evaluated the activity of these enzymes in promastigotes from 23 L. braziliensis isolates as a possible parasite-related factor that could influence the clinical outcome of the disease. Methodology/Principal Findings Our results show that the isolates differ in their ability to hydrolyze adenine nucleotides. Furthermore, we observed a positive correlation between the time for peak of lesion development in C57BL/6J mice and enzymatic activity and clinical manifestation of the isolate. In addition, we found that L. (V.) braziliensis isolates obtained from mucosal lesions hydrolyze higher amounts of adenine nucleotides than isolates obtained from skin lesions. One isolate with high (PPS6m) and another with low (SSF) ecto-nucleotidase activity were chosen for further studies. Mice inoculated with PPS6m show delayed lesion development and present larger parasite loads than animals inoculated with the SSF isolate. In addition, PPS6m modulates the host immune response by inhibiting dendritic cell activation and NO production by activated J774 macrophages. Finally, we observed that the amastigote forms from PPS6m and SSF isolates present low enzymatic activity that does not interfere with NO production and parasite survival in macrophages. Conclusions/Significance Our data suggest that ecto-nucleotidases present on the promastigote forms of the parasite may interfere with the establishment of the immune response with consequent impaired ability to control parasite dissemination and this may be an important factor in determining the clinical outcome of leishmaniasis.

Identification of Virulence Factors in Leishmania infantum Strains by a Proteomic Approach

Knowledge of Leishmania virulence is essential for understanding how the contact between the pathogen and host cells can lead to pathogenesis. Virulence in two L. infantum strains was characterized using macrophages and hamsters. Next, we used difference gel electrophoresis (DIGE) and mass spectrometry to identify the differentially expressed proteins. A total of 63 spots were identified corresponding to 36 proteins; 20 were up-regulated, in which 16 had been previously associated with Leishmania virulence. Considering our results and what has been reported before, we suggest the hypothesis that L. infatum virulence could be a result of the increased expression of KMP-11 and metallopeptidase, associated with an improved parasite-host interacting efficiency and degradation of the protective host proteins and peptides, respectively. Other factors are tryparedoxin peroxidase and peroxidoxin, which protect the parasite against the stress response, and 14-3-3 protein-like, which can prolong infected host cell lifetime. Proteins as chaperones and endoribonuclease L-PSP can increase parasite survival. Enolase is able to perform versatile functions in the cell, acting as a chaperone or in the transcription process, or as a plasminogen receptor or in cell migration events. As expected in more invasive cells with high replication rates, energy consumption and protein synthesis are higher, with up-regulation of Rieske iron-sulfur protein precursor, EF-2, S-adenosylhomocysteine, and phosphomannomutase.

American tegumentary leishmaniasis: effectiveness of an immunohistochemical protocol for the detection of Leishmania in skin.

Background American tegumentary leishmaniasis (ATL) is endemic in Latin America, where Brazil has over 27 thousand cases per year. The aim of the present study was to develop an immunohistochemical method (IHC) for ATL diagnosis. For this purpose, we used serum from a dog naturally infected with Leishmania (L) infantum (canine hyperimmune serum) as the primary antibody, followed by a detection system with a secondary biotinylated antibody. Methodology Skin samples were obtained from 73 patients in an endemic area of Caratinga, Minas Gerais (MG) State, Brazil all testing positive for ATL with the Montenegro skin test, microscopy, and PCR. Canine hyperimmune serum of a dog naturally infected with Leishmania (L.) infantum was employed as a primary antibody in an immunohistochemical diagnostic method using streptavidin-biotin peroxidase. To assess the specificity of this reaction, IHC assays employing two monoclonal antibodies were carried out. As the polymer-based technology is less time-consuming and labor intensive than the IHC labeled streptavidin-biotin peroxidase method, we compared the two methods for all samples. Results The IHC method detected ATL in 67 of the 73 cases (91.8%). Immunolabeled parasites were primarily detected inside macrophages either in the superficial or the deep dermis. Detection was facilitated by the high contrast staining of amastigotes (dark brown) against the light blue background. A lower detection rate (71.2%) was observed with the both of the monoclonal Leishmania antibodies compared to the canine hyperimmune serum. This may have been due to a non-specific background staining observed in all histological samples rendering positive detection more difficult. The higher efficacy of the canine hyperimmune serum in the IHC method was confirmed by the method using streptavidin-biotin peroxidase as well as that with the polymer-based technology (biotin-avidin-free system). Conclusions The data are encouraging with regard to validating IHC as a standard alternative method for ATL diagnosis.

Histopathology of Leishmania major infection: revisiting L. major histopathology in the ear dermis infection model

We describe the relationship between lesion outcome and histopathological hallmarks in susceptible (BALB/c) and resistant (C57BL/6 and IL-4-deficient BALB/c) mouse strains over the course of a 12-week-infection with Leishmania major in the ear. The infiltration of mononuclear cells and polymorphonuclear cells occurred within 6 h and mononuclear cells predominated one week post-infection. Permissive intracellular growth of the pathogen was associated with non-healing lesions. In contrast, tissue damage and clearance of the parasite was observed in healing lesions and was associated with inducible nitric oxide synthase expression. The identification of the structural components of tissue reaction to the parasite in this study furthers our understanding of subjacent immune effector mechanisms.

Short-term protection conferred by Leishvacin® against experimental Leishmania amazonensis infection in C57BL/6 mice

To date, there is no vaccine available against human leishmaniasis. Although some vaccination protocols can induce immunity in murine models, they fail to induce protection in humans. The reasons for that remain unclear. The aim of the present study was to characterize the changes in the pattern of the immune response during subcutaneous vaccination with Leishvacin® in mice. We also investigated whether IFN-γ and nitric oxide synthase are indispensable for the protection elicited by the vaccine. C57BL/6 WT vaccinated mice showed smaller lesions and fewer numbers of parasites in footpads until 8 weeks post-infection. Up to this time, they produced higher levels of IFN-γ, IL-2, IL-4, IL-17A and IL-10 and higher specific antibody response than control non-vaccinated mice. Moreover, we showed that IFN-γ, most likely by induction of iNOS expression, is essential for immunity. However, after 12 weeks of infection, we observed loss of difference in lesion size and parasite burden between the groups. Loss of resistance was associated with the disappearance of differences in cytokine patterns between vaccinated and control mice, but not of antibody response, which remained different until a later time of infection. The reversal of resistance to L. amazonensis could not be explained by upregulation of regulatory cytokines. Our data point to a subversion of the host immune response by L. amazonensis even when a protective response was previously induced.

A potential link among antioxidant enzymes, histopathology and trace elements in canine visceral leishmaniasis

Canine visceral leishmaniasis (CVL) is a severe and fatal systemic chronic inflammatory disease. We investigated the alterations in, and potential associations among, antioxidant enzymes, trace elements and histopathology in CVL. Blood and tissue levels of Cu‐Zn superoxide dismutase, catalase and glutathione peroxidase were measured in mixed‐breed dogs naturally infected with Leishmania infantum chagasi, symptomatic (n = 19) and asymptomatic (n = 11). Serum levels of copper, iron, zinc, selenium and nitric oxide, and plasma lipid peroxidation were measured. Histological and morphometric analyses were conducted of lesions in liver, spleen and lymph nodes. We found lower blood catalase and glutathione peroxidase activity to be correlated with lower iron and selenium respectively. However, higher activity of Cu‐Zn superoxide dismutase was not correlated with the increase in copper and decreased in zinc observed in infected animals compared to controls. Organ tissue was characterized by lower enzyme activity in infected dogs than in controls, but this was not correlated with trace elements. Lipid peroxidation was higher in symptomatic than in asymptomatic and control dogs and was associated with lesions such as chronic inflammatory reaction, congestion, haemosiderin and fibrosis. Systemic iron deposition was observed primarily in the symptomatic dogs showing a higher tissue parasite load. Dogs with symptomatic CVL displayed enhanced LPO and Fe tissue deposition associated with decreased levels of antioxidant enzymes. These results showed new points in the pathology of CVL and might open new treatment perspectives associated with antioxidants and the role of iron in the pathogenesis of CVL.

Determination of cadmium and lead in cassava employing slurry sampling and graphite furnace atomic absorption spectrometry after multivariate optimization

A low cost and efficient method to determine Cd and Pb levels in cassava by graphite furnace atomization atomic absorption spectrometry (GF AAS) employing slurry sampling was developed. Slurries were prepared by cryogenic grinding of samples (40 mg), sieving for grain size at 50 mesh and addition of 2% v/v nitric acid and 1% v/v hydrogenous peroxide up to 2 mL of volume. The slurry was maintained homogeneous using air bubbling by an aquarium pump. Tungsten (500 μg) and titanium (500 μg) were the best permanent modifiers for Cd and Pb, respectively. The parameters of merit obtained under optimized conditions, AT (atomization temperature) = 1400 °C, PT (pyrolysis temperature) = 600 °C, pt (pyrolysis time) = 20 s and permanent W (500 μg) for Cd; AT = 1400 °C, PT = 450 °C, pt = 20 s, and Ti (500 μg) for Pb were: linear working range up to 6 and 50 μg L−1 for Cd and Pb, respectively; limit of detection (0.001 and 0.02) μg g−1 for Cd and Pb, respectively; and aqueous calibration, with r2 > 0.99. The accuracy was evaluated by recovery tests and by analysis of certified reference material (NIST – SRM 1568a, rice flour) and was in agreement with the certified values for the two analytes: 0.021 ± 0.002 (n = 5 replicates) for Cd (certified value 0.022 ± 0.002) and <LOQ (n = 5) for Pb (certified value <0.010). The average sample concentrations (n = 3) were between <LOQ to 5.8 ng g−1 and 0.08 to 3.18 μg g−1 for Cd and Pb, respectively.

Effectiveness of an immunohistochemical protocol for Leishmania detection in different clinical forms of American tegumentary leishmaniasis

American tegumentary leishmaniasis (ATL) is a neglected disease widely distributed in Latin America. In Brazil, it is caused by different Leishmania species belonging to the Subgenera Viannia and Leishmania. ATL diagnosis is routinely based on clinical, epidemiological, parasitological and immunological (delayed-type hypersensitivity skin test-DTH) evidences. The main objective of this work was to determine the efficacy of a previous immunohistochemical (IHC) method developed by our group. Seventy eight skin biopsies from patients with different ATL clinical forms and origins were evaluated. The method was previously standardized in ATL patients from the municipality of Caratinga, Minas Gerais, Brazil, all infected with Leishmania (V.) braziliensis. Here, it is evaluated in patients from the North, Southeast and Midwest regions of Brazil. Clinical, parasitological (biopsy PCR) and immunological (Montenegro skin test-MST) diagnosis were performed prior to IHC procedure. The IHC procedure detected 70.5% of the cases having a high agreement with MST diagnosis (kappa=0.84). A distinguished contribution of this work is that IHC succeed in diagnosing some negative DTH patients. Those were infected with Leishmania (L.) amazonensis, commonly causing the anergic form of the disease. In conclusion, IHC succeed in detecting ATL caused by different Leishmania species from various geographic regions and clinical status. Although it was not able to detect ATL in all patients, it was better than MST providing an additional tool for the diagnosis of ATL patients. There was no significant correlation between clinical forms and histological features including the presence of necrosis.

Glycol Methacrylate Embedding for the Histochemical Study of the Gastrointestinal Tract of Dogs Naturally Infected with Leishmania Infantum

In canine visceral leishmaniasis a diffuse chronic inflammatory exudate and an intense parasite load throughout the gastrointestinal tract (GIT) has been previously reported. However, these studies did not allow a properly description of canine cellular morphology details. The aim of our study was to better characterize these cells in carrying out a qualitative and quantitative histological study in the gastrointestinal tract of dogs naturally infected with Leishmania infantum by examining gut tissues embedded in glycol methacrylate. Twelve infected adult dogs were classified in asymptomatic and symptomatic. Five uninfected dogs were used as controls. After necropsy, three samples of each gut segment, including oesophagus, stomach, duodenum, jejunum, ileum, cecum, colon, and rectum were collected and fixed in Carnoy’s solution for glycol methacrylate protocols. Sections were stained with hematoxylin-eosin, toluidine blue borate, and periodic acid-Schiff stain. Leishmania amastigotes were detected by immunohistochemistry employed in both glycol methacrylate and paraffin embedded tissues. The quantitative histological analysis showed higher numbers of plasma cells, lymphocytes and macrophages in lamina propria of all segments of GIT of infected dogs compared with controls. The parasite load was more intense and cecum and colon, independently of the clinical status of these dogs. Importantly, glycol methacrylate embedded tissue stained with toluidine blue borate clearly revealed mast cell morphology, even after mast cell degranulation. Infected dogs showed lower numbers of mast cells in all gut segments than controls. Despite the glycol methacrylate (GMA) protocol requires more attention and care than the conventional paraffin processing, this embedding procedure proved to be especially suitable for the present histological study, where it allowed to preserve and observe cell morphology in fine detail.

The endogenous cytokine profile and nerve fibre density in mouse ear Leishmania major-induced lesions related to nociceptive thresholds

Several reports have shown that cutaneous leishmaniasis lesions are painless, suggesting that Leishmania infection interferes with pain perception. Comparisons of inflammation-induced hyperalgesia between BALB/c and C57BL/6 mice have been little explored in the literature, and comparative data regarding nociception in leishmaniasis are non-existent. In susceptible BALB/c mice and resistant C57BL/6 mice that were intradermally inoculated with a low dose of Leishmania major in the ear, we investigated the variation in nociception over a 12-wk period post-infection and this variations association with the structure of nerve fibres and the presence of endogenous cytokines that are classically considered hyper- or hypo-nociceptive. Infected BALB/c mice presented susceptibility and severe lesions. Infected C57BL/6 mice exhibited resistance and healing lesions. The immune response involved pro- and anti-inflammatory cytokine secretion, respectively. The infection-induced hypoalgesia in BALB/c mice after wks 9 was accompanied by decreased levels of IL-6 and IL-10 in ear tissue with intact nerves. C57BL/6 mice showed short-lived hyperalgesia in wks 2, which was related to increased local levels of IL-6, KC/CXCL-1, TNF-α and IL-10 and a decrease in nerve density. The increase in pro-inflammatory cytokine IL-6, KC/CXCL-1 and TNF-α levels during hyperalgesia suggested a role for these mediators in afferent nerve sensitisation, which was secondary to the inflammatory damage of nerve fibres stained by PGP 9.5. In contrast, the mechanisms of hypoalgesia may include the downregulation of cytokines, the preservation of the structure of nerve endings, and as yet uninvestigated unidentified differences in neurotransmitter release or a direct role of the parasites in the context of the progressive and permissive inflammatory response of BALB/c mice.