Vitamin d and leishmaniasis: Neither seasonal nor risk factor in canine host but potential adjuvant treatment through cbd103 expression.

Vitamin D (VitD) deficiency has been shown to be a risk factor for a plethora of disorders. We have shown that dogs with clinical leishmaniasis presented lower VitD serum levels than non-infected dogs, and even lower than those with asymptomatic infection. However, if VitD deficiency is a risk factor to develop clinical leishmaniasis remains to be answered. It is also unknown if VitD participates in Leishmania control. First, we retrospectively analysed VitD concentration in serum samples from 36 healthy dogs collected in different periods of the year concluding that there isn't a seasonal variation of this vitamin in dogs. We also included 9 dogs with clinical leishmaniasis and 10 non-infected healthy dogs, in which we measured VitD levels at the beginning of the study, when all dogs were negative for serology and qPCR, and 1 year later. Whereas non-infected dogs showed no change in VitD levels along the study, those developing clinical leishmaniasis showed a significant VitD reduction at the end of the study (35%). When we compared VitD concentration between the two groups at the beginning of the study, no differences were detected (43.6 (38-59) ng/mL, P = 0.962). Furthermore, an in vitro model using a canine macrophage cell line proved that adding active VitD leads to a significant reduction in L. infantum load (31.4%). Analyzing expression of genes related to VitD pathway on primary canine monocytes, we showed that CBD103 expression was significantly enhanced after 1,25(OH)2D addition. Our results show that VitD concentration is neither seasonal nor a risk factor for developing canine leishmaniasis, but it diminishes with the onset of clinical disease suggesting a role in parasitic control. Our in vitro results corroborate this hypothesis and point out that VitD regulates infection through CBD103 expression. These results open the possibility for studies testing VitD as an adjuvant in leishmaniasis therapy.

Pre-clinical evidences of the antileishmanial effects of diselenides and selenocyanates.

A series of thirty-one selenocompounds covering a wide chemical space was assessed for in vitro leishmanicidal activities against Leishmania infantum amastigotes. The cytotoxicity of those compounds was also evaluated on human THP-1 cells. Interestingly most tested derivatives were active in the low micromolar range and seven of them (A.I.3, A.I.7, B.I.1, B.I.2, C.I.7 C.I.8 and C.II.8) stood out for selectivity indexes higher than the ones exhibited by reference compounds mitelfosine and edelfosine. These leader compounds were evaluated against infected macrophages and their trypanothione reductase (TryR) inhibition potency was measured to further approach the mechanism by which they caused their action. Among them diselenide tested structures were pointed out for their ability to reduce infection rates. Three of the leader compounds inhibited TryR effectively, therefore this enzyme may be implicated in the mechanism of action by which these compounds cause their leishmanicidal effect.

Controlling phlebotomine sand flies to prevent canine Leishmania infantum infection: A case of knowing your enemy.

Leishmaniosis caused by Leishmania infantum is a widespread zoonotic disease that can be transmitted to animals and humans by their vectors, blood-sucking phlebotomine sand flies. To prevent canine leishmaniosis across the whole Mediterranean region, vector control is essential. Because of phlebotomine breeding sites are diverse, environmental larval controls have limited practical value. Control methods of adults are being evaluated, such as selective baits based on sugar feeding of males and females or Attractive Toxic Sugar Baits (ATSB), and the indoor use of Long-Lasting-Insecticidal Nets (LLINs) treated with permethrin to prevent sand fly bites complementing the Indoor Residual Spraying (IRS) approach suggested by WHO. Although several strategies exist, the best control measure to prevent canine Leishmania infantum is to treat dogs using biocidal topical formulations based on legal insecticides (PTs18) or repellents (PTs19) (as collars, spot-ons and/or sprays) during the period when the vectors are active. This means we need to really know the biology and life cycle of the sand fly vector. According to available data, by mapping ambient temperatures we can already predict high risk areas where vector densities will be higher. In ongoing research, new candidates are emerging to fight against sand flies including natural plant extracts with low impacts on the environment and host animal. Other options in the future could be systemic insecticides to help reduce sand fly populations in high density areas. In parallel, health authorities and professionals involved in animal and public health (veterinarians, physicians, entomologists and epidemiologists) must work together in a One Health approach to minimize Leishmania infection. Veterinarians play a crucial role in liaising between key stake holders and dog owners to ensure the latter act responsibly in using repellents as a preventive measure against sand fly bites.

In vitro antileishmanial effects of Physalis angulata root extract on Leishmania infantum.

OBJECTIVE: In the present study, we evaluated the effects of the aqueous extract of Physalis angulata root (AEPa) on Leishmania infantum proliferation, morphology, and the driving mechanism in leishmanicidal activity and modulatory action on macrophages. METHODS: L. infantum promastigotes were treated with 50 and 100 µg/mL AEPa for 72 h and then antipromastigote assay was performed by counts in a Newbauer chamber, morphological changes were analyzed by transmission electron microscopy and the mechanism of the leishmanicidal activity was detected. In addition, macrophages were infected with L. infantum and were used to evaluate anti-amastigote activity of AEPa and effects of AEPa on cytokine secretion after 72-hour treatment. RESULTS: Treatment with AEPa reduced the numbers of L. infantum promastigotes (50% inhibitory concentration (IC50) = 65.9 µg/mL; selectivity index (SI) = 22.1) and amastigotes (IC50 = 37.9 µg/mL; SI = 38.5) compared with the untreated control. Amphotericin B reduced 100% of the promastigote numbers after 72 h of treatment (IC50 = 0.2 µg/mL). AEPa induced several morphological changes and increased the production of reactive oxygen species and apoptotic death in promastigotes after treating for 72 h. AEPa (100 µg/mL) promoted tumor necrosis factor-α secretion in macrophages infected with L. infantum after 72 h of treatment, but did not induce an increase in this cytokine in noninfected macrophages. In addition, AEPa showed no cytotoxic effect on J774-A1 cells (50% cytotoxic concentration >1000 µg/mL). CONCLUSION: AEPa presented antileishmanial activity against the promastigotes and amastigotes of L. infantum without macrophage cytotoxicity; these results show that natural products such as P. angulata have leishmanicidal potential and in the future may be an alternative treatment for leishmaniasis.

In Vitro antileishmania activity of sesquiterpene-rich essential oils from Nectandra species.

CONTEXT: New antileishmanias are needed because of toxicity, high cost and resistance problems associated with available drugs. Nectandra (Lauraceae) produces several classes of compounds but its essential oil has not previously been reported to have antileishmania activity. OBJECTIVE: We evaluated the cytotoxicity and antileishmania activity of essential oils from Nectandra amazonum Nees, N. gardneri Meisn., N. hihua (Ruiz & Pav.) Rohwer and N. megapotamica (Spreng.) Mez. MATERIALS AND METHODS: Nectandra oils were extracted from stem bark/leaves by hydrodistillation and compounds were identified by GC-MS. Oils were tested against Leishmania infantum and L. amazonensis intracellular amastigotes and nitric oxide production was evaluated. Cytotoxicity was achieved on NIH/3T3 and J774.A1 cells for the selectivity index (SI). RESULTS AND DISCUSSION: Nectandra gardneri was active against L. infantum and L. amazonensis (IC50 = 2.7 ± 1.3/2.1 ± 1.06 µg/mL) and contained 85.4% sesquiterpenes, of which 58.2% was intermediol. Besides low cytotoxicity (SI >11.3), N. gardneri induced a significant increase in NO production by L. infantum-infected macrophages. Nectandra hihua had the best activity on L. infantum amastigotes (IC50 = 0.2 ± 1.1 µg/mL). This oil was 89.0% sesquiterpenes, with 28.1% bicyclogermacrene. The two specimens of N. megapotamica had different activities on amastigotes. The one richer in sesquiterpenes (49.9%) was active against both species (IC50 = 12.5 ± 1.4/21.3 ± 1.2) and had phenylpropanoid E-asarone as the main compound (42.4%). Nectandra amazonum showed moderate activity on both the species (IC50 = 31.9 ± 2.0/22.1 ± 1.3 µg/mL) and low selectivity (0.9 < SI >2.6), probably due to the major presence of ß-caryophyllene (28.5%). CONCLUSIONS: Our data identify compounds that can now be isolated and used for the development of new antileishmanias.

Antiprotozoal activity of extracts and isolated triterpenoids of 'carnauba' (Copernicia prunifera) wax from Brazil.

CONTEXT: 'Carnauba' wax is a natural product obtained from the processing of the powder exuded from Copernicia prunifera (Miller) H. E. Moore (Arecaceae). This material is widely used in the Brazilian folk medicine, including the treatment of rheumatism and syphilis. OBJECTIVE: To investigate the antiprotozoal activity of hexane and EtOH extracts from the 'carnauba' wax as well as from the isolated compounds from the bioactive extracts. MATERIAL AND METHODS: Two different samples of 'carnauba' (C. prunifera) waxes - types 1 and 4 - were individually extracted using hexane (EH) and EtOH (EE). Aliquots of hexane (type 1 - EH-1 and EH-4) and EtOH (type 4 - EE-1 and EE-4) extracts were tested against promastigote (2-200 µg/mL in DMSO during 48 h at 24 °C) and amastigote (3-150 µg/mL in DMSO during 120 h at 37 °C) forms of Leishmania infantum as well as against trypomastigote (3-150 µg/mL in DMSO during 24 h at 37 °C) forms of Trypanosoma cruzi. Bioactive extracts EH-1 and EE-4 were subjected to a bioactivity-guided fractionation to afford three dammarane-type triterpenoids (1-3). The in vitro antiprotozoal activities of the obtained compounds were evaluated as described above. Additionally, the cytotoxicity activity of compounds 1-3 against mammalian conjunctive cells (NCTC - 2-200 µg/mL in DMSO during 48 h at 37 °C) was determined. RESULTS: From the bioactive hexane and EtOH extracts from the 'carnauba' (C. prunifera) wax, were isolated three dammarane-type triterpenoids: (24R*)-methyldammar-25-ene-3ß,20-diol (carnaubadiol, 1), (24R*)-methyldammara-20,25-dien-3-one (2) and (24R*)-methyldammara-20,25-dien-3α-ol (3). These compounds were identified based on the analysis of NMR and MS spectroscopic data. Compounds 1-3 were effective against the intracellular amastigotes of L. infantum, with IC50 values ranging from 8 to 52 µM, while compounds 1 and 3 displayed activity against trypomastigote forms of T. cruzi with IC50 values of 15 and 35 µM, respectively. The mammalian cytotoxicity assay demonstrated no damage to NCTC conjunctive cells up to 200 µM, except for compound 1, which demonstrated a CC50 value of 34 µM. CONCLUSION: Based on the results, it was possible to conclude that the detected antiprotozoal bioactivity of 'carnauba' (C. prunifera) wax extracts could be related to the presence of the natural dammarane triterpenoid derivatives. The results suggested that these compounds could be used as promising scaffolds for drug design studies for leishmaniasis and Chagas disease.

Antileishmanial activity and evaluation of the mechanism of action of strychnobiflavone flavonoid isolated from Strychnos pseudoquina against Leishmania infantum.

The present study aimed to investigate the in vitro antileishmanial activity of strychnobiflavone flavonoid against Leishmania infantum, as well as its mechanism of action, and evaluate the ex vivo biodistribution profile of the flavonoid in naive BALB/c mice. The antileishmanial activity (IC50 value) of strychnobiflavone against stationary promastigote and amastigote-like stages of the parasites was of 5.4 and 18.9 µM, respectively; with a 50% cytotoxic concentration (CC50) value of 125.0 µM on murine macrophages, resulting in selectivity index (SI) of 23.2 and 6.6, respectively. Amphotericin B, used as a positive control, presented SI values of 7.6 and 3.3 for promastigote and amastigote-like stages of L. infantum, respectively. The strychnobiflavone was also effective in reducing in significant levels the percentage of infected macrophages, as well as the number of amastigotes per macrophage, after the treatment of infected macrophages using the flavonoid. By using different fluorescent probes, we investigated the bioenergetics metabolism of L. infantum promastigotes and demonstrated that the flavonoid caused the depolarization of the mitochondrial membrane potential, without affecting the production of reactive oxygen species. In addition, using SYTOX(®) green as a fluorescent probe, the strychnobiflavone demonstrated no interference in plasma membrane permeability. For the ex vivo biodistribution assays, the flavonoid was labeled with technetium-(99m) and studied in a mouse model by intraperitoneal route. After a single dose administration, the scintigraphic images demonstrated a highest uptake by the liver and spleen of the animals within 60 min, resulting in low concentrations after 24 h. The present study therefore demonstrated, for the first time, the antileishmanial activity of the strychnobiflavone against L. infantum, and suggests that the mitochondria of the parasites may be the possible target organelle. The preferential distribution of this compound into the liver and spleen of the animals could warrant its employ in the treatment of visceral leishmaniasis.

Leishmanicidal and cytotoxic activities of Nigella sativa and its active principle, thymoquinone.

CONTEXT: Leishmaniasis is a complex disease with a broad spectrum of clinical presentations. OBJECTIVE: We evaluated the anti-leishmanial effects of Nigella sativa L. (Ranunculaceae) against Leishmania tropica and Leishmania infantum with an in vitro model. MATERIALS AND METHODS: Antileishmanial effects of essential oil and methanolic extract of N. sativa (0-200 µg/mL) and thymoquinone (0-25 µg/mL) on promastigotes of both species and their cytotoxicity activities against murine macrophages were evaluated using the MTT assay at 24, 48, and 72 h. Moreover, their leishmanicidal effects against amastigotes were investigated in a macrophage model, for 48 and 72 h. RESULTS: The findings showed that essential oil (L. tropica IC50 9.3 µg/mL and L. infantum IC50 11.7 µg/mL) and methanolic extract (L. tropica IC50 14.8 µg/mL and L. infantum IC50 15.7 µg/mL) of N. sativa, particularly thymoquinone (L. tropica IC50 1.16 µg/mL and L. infantum IC50 1.47 µg/mL), had potent antileishmanial activity on promastigotes of both species after 72 h. In addition, essential oil (L. tropica IC50 21.4 µg/mL and L. infantum IC50 26.3 µg/mL), methanolic extract (L. tropica IC50 30.8 µg/mL and L. infantum IC50 34.6 µg/mL), and thymoquinone (L. tropica IC50 2.1 µg/mL and L. infantum IC50 2.6 µg/mL) mediated a significant decrease in the growth rate of amastigote forms of both species. Thymoquinone (CC50 38.8 µg/mL) exhibited higher cytotoxic effects against murine macrophages than the other extracts. CONCLUSION: N. sativa, especially its active principle, thymoquinone, showed a potent leishmanicidal activity against L. tropica and L.infantum with an in vitro model.

Histamine H1-receptor antagonists against Leishmania (L.) infantum: an in vitro and in vivo evaluation using phosphatidylserine-liposomes.

Considering the limited and toxic therapeutic arsenal available for visceral leishmaniasis (VL), the drug repositioning approach could represent a promising tool to the introduction of alternative therapies. Histamine H1-receptor antagonists are drugs belonging to different therapeutic classes, including antiallergics and anxyolitics. In this work, we described for the first time the activity of H1-antagonists against L. (L.) infantum and their potential effectiveness in an experimental hamster model. The evaluation against promastigotes demonstrated that chlorpheniramine, cinnarizine, hydroxyzine, ketotifen, loratadine, quetiapine and risperidone exerted a leishmanicidal effect against promastigotes, with IC50 values in the range of 13-84µM. The antihistaminic drug cinnarizine demonstrated effectiveness against the intracellular amastigotes, with an IC50 value of 21µM. The mammalian cytotoxicity was investigated in NCTC cells, resulting in IC50 values in the range of 57-229µM. Cinnarizine was in vivo studied as a free formulation and entrapped into phosphatidylserine-liposomes. The free drug was administered for eight consecutive days at 50mg/kg by intraperitoneal route (i.p.) and at 100mg/kg by oral route to L. infantum-infected hamsters, but showed lack of effectiveness in both regimens, as detected by real time PCR. The liposomal formulation was administered by i.p. route at 3mg/kg for eight days and reduced the parasite burden to 54% in liver when compared to untreated group; no improvement was observed in the spleen of infected hamsters. Cinnarizine is the first antihistaminic drug with antileishmanial activity and could be used as scaffold for drug design studies for VL.

Morphological and physiological changes in Leishmania promastigotes induced by yangambin, a lignan obtained from Ocotea duckei.

We have previously demonstrated that yangambin, a lignan obtained from Ocotea duckei Vattimo (Lauraceae), shows antileishmanial activity against promastigote forms of Leishmania chagasi and Leishmania amazonensis. The aim of this study was to determine the in vitro effects of yangambin against these parasites using electron and confocal microscopy. L. chagasi and L. amazonensis promastigotes were incubated respectively with 50 µg/mL and 65 µg/mL of pure yangambin and stained with acridine orange. Treated-parasites showed significant alterations in fluorescence emission pattern and cell morphology when compared with control cells, including the appearance of abnormal round-shaped cells, loss of cell motility, nuclear pyknosis, cytoplasm acidification and increased number of acidic vesicular organelles (AVOs), suggesting important physiological changes. Ultrastructural analysis of treated-promatigotes showed characteristics of cell death by apoptosis as well as by autophagy. The presence of parasites exhibiting multiples nuclei suggests that yangambin may also affect the microtubule dynamic in both Leishmania species. Taken together our results show that yangambin is a promising agent against Leishmania.

Effects of essential oils from Cymbopogon citratus (DC) Stapf., Lippia sidoides Cham., and Ocimum gratissimum L. on growth and ultrastructure of Leishmania chagasi promastigotes.

The current therapy for leishmaniasis, which affects annually about 2 million people, is far from satisfactory. All available drugs require parenteral administration and are potentially toxic. Plant essential oils have been traditionally used in folk medicine and appear as valuable alternative source for chemotherapeutic compounds. In this study, we demonstrated the effect of essential oils from Cymbopogon citratus, Lippia sidoides, and Ocimum gratissimum on growth and ultrastructure of Leishmania chagasi promastigote forms. Steam distillation was used to isolate the essential oils, and their constituents were characterized by gas chromatography coupled to mass spectrometry and nuclear magnetic resonance. All essential oils showed in vitro inhibitory action on L. chagasi promastigotes growth in a dose-dependent way, with IC(50)/72 h of 45, 89, and 75 microg/mL for C. citratus, L. sidoides, and O. gratissimum, respectively. Drastic morphological alterations were observed in all essential oil-treated parasites, including cell swelling, accumulation of lipid droplets in the cytoplasm, and increase of acidocalcisome volume. Furthermore, aberrant-shaped cells with multi-septate body were observed by scanning electron microscopy, suggesting an additional effect on cytokinesis. Taken together, our data show that these essential oils affect the parasite viability being the C. citratus essential oil the most effective against L. chagasi.