Raíssa Bocchi Pedroso

Leishmanicidal activity of synthetic chalcones in Leishmania (Viannia) braziliensis

The treatment of American cutaneous leishmaniasis (ACL) is based on a small group of compounds that were developed decades ago, all of which are highly toxic and have a high rate of treatment failure. The chalcones show leishmanicidal activity, yet few studies have evaluated this activity against Leishmania (Viannia) braziliensis, one of the most important species of Leishmania across Latin America. Four new synthetic chalcones (1-4) were evaluated for inhibitory activity in vitro against promastigotes and intracellular parasites 24h post infection of L. (V.) braziliensis, cytotoxicity for macrophages J774.A1 and red blood cells, and the ability to stimulate nitric oxide production. The results for the inhibitory concentration for 50% of the promastigotes (IC50) (1.38±1.09-6.36±2.04μM), cytotoxic concentration for 50% of the macrophages (CC50) (13.49±3.13-199.43±4.11μM), and selectivity index (SI) (3.76 to 33.94) indicate that all chalcones (1-4) showed an effect on promastigotes of L. (V.) braziliensis; chalcone 2 had the highest SI. The haemolytic assay with chalcones 1 (301.93μM), 2 (534.18μM), 3 (419.46μM) and 4 (381.11μM) showed 0.00%, 2.33%, 0.57% and 1.74% haemolysis, respectively. All chalcones significantly reduced the infection index of macrophages by parasites; for chalcones (1-3) this effect may be dependent on nitric-oxide production by macrophages. The chalcones tested exhibited inhibitory activity for promastigotes and intracellular parasites of L. (V.) braziliensis, with low toxicity for macrophages and red blood cells. The anti-Leishmania activity of chalcones (1-3) may depend on the stimulation of nitric-oxide production in the initial stage of infection. These results show an initially encouraging potential for the use of chalcones (1-4) to treat ACL.

Evaluation of the reference value for the Montenegro skin test

INTRODUCTION The Montenegro skin test (MST) has good clinical applicability and low cost for the diagnosis of American tegumentary leishmaniasis (ATL). However, no studies have validated the reference value (5mm) typically used to discriminate positive and negative results. We investigated MST results and evaluated its performance using different cut-off points. METHODS The results of laboratory tests for 4,256 patients with suspected ATL were analyzed, and 1,182 individuals were found to fulfill the established criteria. Two groups were formed. The positive cutaneous leishmaniasis (PCL) group included patients with skin lesions and positive direct search for parasites (DS) results. The negative cutaneous leishmaniasis (NCL) group included patients with skin lesions with evolution up to 2 months, negative DS results, and negative indirect immunofluorescence assay results who were residents of urban areas that were reported to be probable sites of infection at domiciles and peridomiciles. RESULTS The PCL and NCL groups included 769 and 413 individuals, respectively. The mean ± standard deviation MST in the PCL group was 12.62 ± 5.91mm [95% confidence interval (CI): 12.20-13.04], and that in the NCL group was 1.43 ± 2.17mm (95% CI: 1.23-1.63). Receiver-operating characteristic curve analysis indicated 97.4% sensitivity and 93.9% specificity for a cut-off of 5mm and 95.8% sensitivity and 97.1% specificity for a cut-off of 6mm. CONCLUSIONS Either 5mm or 6mm could be used as the cut-off value for diagnosing ATL, as both values had high sensitivity and specificity.

Immune Responses in Broiler Chicks Fed Propolis Extraction Residue-supplemented Diets.

This study was conducted to evaluate the effect of inclusion of propolis extraction residue in the feed of broilers from 1 to 21 d of age on phagocytic activity of macrophages, cutaneous basophil hypersensitivity response to phytohemagglutinin, antibody production against Newcastle disease, lymphoid organ weight and hematological profile and to determine the optimal level of inclusion. 120 chicks, reared in metabolism cages until 21 days of age, were distributed in a completely randomized design, with five treatments (0%, 1%, 2%, 3%, and 4% of propolis residue) and six replications. The relative weight of thymus and monocyte percentage were affected by propolis residue, with a quadratic response (p<0.05) and lowest values estimated at 2.38% and 2.49%, respectively. Changes in relative weight of cloacal bursa and spleen, percentage of lymphocyte, heterophil, basophil, eosinophil, and heterophil:lymphocyte ratio, antibody production against Newcastle disease, phagocytic activity of macrophages and the average number of phagocytosed erythrocytes were not observed. The nitric oxide production with regard to positive control (macrophages+erythrocytes) decreased linearly (p<0.05) with increased doses of propolis residue. The remaining variables of nitric oxide production (negative control – macrophages, and difference between the controls) were not affected by propolis residue. The cutaneous basophil hypersensitivity response to phytohemagglutinin as determined by the increase in interdigital skin thickness exhibited a quadratic response (p<0.05), which predicted a lower reaction response at a dose of 2.60% of propolis residue and highest reaction response after 43.05 hours of phytohemagglutinin injection. The inclusion of 1% to 4% of propolis extraction residue in broiler diets from 1 to 21 days of age was not able to improve the immune parameters, despite the modest changes in the relative weight in thymus, blood monocyte percentage, nitric oxide concentration, and interdigital reaction to phytohemagglutinin.

Antileishmanial activity of the essential oil from Tetradenia riparia obtained in different seasons

The herbaceous shrub Tetradenia riparia has been traditionally used to treat inflammatory and infectious diseases. Recently, a study showed that T. riparia essential oil (TrEO) obtained in summer has antileishmanial effects, although these results could be influenced by seasonal variation. This study evaluated the activity of the TrEO obtained in different seasons against Leishmania (Leishmania) amazonensis, in vitro and in vivo. The compounds in the TrEO were analysed by gas chromatography-mass spectrometry; terpenoids were present and oxygenated sesquiterpenes were the majority compounds (55.28%). The cytotoxicity and nitric oxide (NO) production were also tested after TrEO treatment. The TrEO from all seasons showed a 50% growth inhibitory concentration for promastigotes of about 15 ng/mL; at 30 ng/mL and 3 ng/mL, the TrEO reduced intracellular amastigote infection, independently of season. The TrEO from plants harvested in summer had the highest 50% cytotoxic concentration, 1,476 ng/mL for J774.A1 macrophages, and in spring (90.94 ng/mL) for murine macrophages. NO production did not change in samples of the TrEO from different seasons. The antileishmanial effect in vivo consisted of a reduction of the parasite load in the spleen. These results suggest that the TrEO has potential effects on L. (L.) amazonensis, consonant with its traditional use to treat parasitic diseases.

Diagnosis of American cutaneous leishmaniasis by enzyme immunoassay using membrane antigens of Leishmania (Viannia) braziliensis.

This study evaluated the reactivity of membrane antigens of Leishmania (Viannia) braziliensis for the diagnosis of ACL by enzyme immunoassay (EIA). Promastigotes of L. (V.) braziliensis were grown in medium 199 and lysed in a sonicator. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting showed that specific proteins of L. (V.) braziliensis (apparent molecular weights 36 kDa and 48-56 kDa) were recognized by sera from ACL patients. These proteins were eluted from the SDS-PAGE and tested in EIA-IgG with sera from ACL patients, healthy individuals, patients with toxoplasmosis, paracoccidioidomycosis, syphilis, tuberculosis, leprosy, and Chagas disease. The EIA-IgG with membrane antigens allowed us to distinguish patients with ACL from healthy individuals and patients with other diseases (P < 0.0001), and showed a sensitivity of 93.3% and specificity of 90.8%, not including Chagas disease patients. 2D-SDS-PAGE followed by Western blotting was performed to improve the characterization of the antigens, and showed a component with isoelectric points near the acid pH side and apparent molecular weights of 48-56 kDa. The results showed good sensitivity and specificity of EIA-IgG with membrane antigens, indicating their potential use for diagnosis of ACL, as well as seroepidemiological surveys and follow-up of clinically cured patients.

Calophyllum brasiliense Modulates the Immune Response and Promotes Leishmania amazonensis Intracellular Death

Calophyllum brasiliense is a plant from the Brazilian rain forests and has been used in folk medicine for the treatment of various diseases, including leishmaniasis. This infectious disease depends on the Leishmania sp. and the host immune response. C. brasiliense antileishmanial activity is well known, but the effects on immune response remain to be investigated. This study showed the leishmanicidal and immunomodulatory effects of a 30 μg/mL of hydroalcoholic extract of C. brasiliense in murine macrophages before and after Leishmania (Leishmania) amazonensis infection. The semiquantitative cytokine RNA expression was determined by RT-PCR and the anti-Leishmania activity was measured by infection index (IF). Hydroalcoholic extract of C. brasiliense reduced more than 95% of IF when used before and after Leishmania infection, with 3 and 24 h of treatment (p < 0.05). C. brasiliense inhibited or reduced significantly (p < 0.05) the TNF-α, IL-1β, IL-18, and IL-10 mRNA expression. The antileishmanial and anti-inflammatory effects showed the potential of C. brasiliense as an alternative therapy for leishmaniasis and it must be investigated.

Detection of Leishmania (Viannia) DNA in leucocytes from the blood of patients with cutaneous leishmaniasis.

INTRODUCTION Cutaneous leishmaniasis (CL) is a serious and global public health issue, with the potential of developing a mucosal form, occurring as subclinical cases, and showing recurrence despite previous treatment. METHODS Polymorphonuclear and mononuclear DNA obtained from 49 patients was subjected to polymerase chain reaction for detection of Leishmania (Viannia). RESULTS DNA was detected in mononuclear cells from two patients with active primary lesions positive for CL, with infection periods of 3 and 6 months, respectively. CONCLUSIONS The DNA of Leishmania (Viannia) indicates probable parasite dissemination possibly explaining subclinical case emergence, lesion recurrence, and mucosal lesion appearance.

AMERICAN CUTANEOUS LEISHMANIASIS WITH UNUSUAL CLINICAL PRESENTATION AND RESPONSE TO TREATMENT

The clinical manifestations and prognosis of cutaneous leishmaniasis (CL) can be influenced by the immune response of the patient and the species of the parasite. A case of atypical clinical presentation of CL, with development of non-characteristic lesions, poor response to therapy, and a long time to resolution is reported. Confirmatory laboratory tests included parasite detection, indirect immunofluorescence, Montenegro skin test, polymerase chain reaction, and parasite identification by multilocus enzyme electrophoresis. The parasite was identified as Leishmaniabraziliensis. The lesion was unresponsive to three complete courses of N-methylglucamine antimoniate intramuscular, and to treatment with pentamidine. The patient did not tolerate amphotericin B. The lesion finally receded after treatment with intravenous N-methylglucamine antimoniate. It is essential to ensure the accuracy of diagnosis and the appropriate treatment, which can include the use a second choice drug or a different route of administration.

AVALIAÇÃO DE PRIMERS ESPECÍFICOS PARA IDENTIFICAÇÃO DE ESPÉCIES DE Leishmania (V.) braziliensis

Leishmaniases are infectious diseases with different clinical forms and prognoses, making accurate species identification particularly important. We evaluated the performance of LBF1/LBR1 L. (V.) braziliensis specific primers by PCR and compared the results with Leishmania spp. identified by monoclonal antibodies (mAbs). Of 29 L. (V.) braziliensis identified by mAbs, 16 (53.3%) were detected; and 7 (63.6%) of 11 unidentified Leishmania spp. showed the 536 bp band. 87.7% of serodeme III Leishmania isolates were identified by these primers. These results indicate a poor correlation between the two identification methods used, and also suggest the existence of genetic variability among L. (V.) braziliensis isolates from the northwest region of Paraná state.