The potential role and apoptotic profile of three medicinal plant extracts on Leishmania tropica by MTT assay, macrophage model and flow cytometry analysis.

INTRODUCTION: Treatment of leishmaniasis with conventional synthetic drugs is a major global challenge. This study was designed to explore the leishmanicidal activity and apoptotic profile of three leaf extracts on Leishmania tropica stages. METHODS: The plants of Quercus velutina, Calotropis procera and Nicotiana tabacum were gathered from Anbarabbad county, in the southeastern part of Kerman province and extracted by maceration method using methanol alcohol. Various concentrations of the extracts (1, 10, 100 and 1000 µg/mL) were used against L. tropica stages to evaluate the inhibitory effect by colorimetric assay, macrophage model and flow cytometry. The MTT assay was conducted to determine the IC50 and CC50 values in promastigotes and J774-A1 macrophages, respectively. For intra-macrophage amastigotes, the leishmanicidal activity was evaluated by calculating the mean number of amastigotes in each macrophage and also IC50 values. The promastigote or amastigote stages with no drug and complete medium without organisms were considered as positive and negative controls, respectively. Meglumine antimoniate (Glucantime) was also used as standard drug. Also, annexin V was used to assess the apoptotic profile. All treatment settings were incubated for a standard time of 72 h in triplicates. Data were analyzed by t-test and ANOVA. RESULTS: The findings showed that all plant extracts inhibited the proliferation rate of promastigotes and amastigotes (P ˂ 0.001); especially, Q. velutina represented the lowest IC50 in both stages. Besides, Q. velutina showed the least number of amastigotes in each macrophage compared to the other groups (4.5 µg/mL). The percentage of parasitic apoptosis at 1000 µg/mL of Q. velutina, C. procera, N. tabacum and Glucantime® were 37.4, 18.6, 8.5 and 52.4, respectively. Amastigotes (clinical stage) were significantly more susceptible to extracts and also Glucantime® than promastigotes (P < 0.001). CONCLUSIONS: This study revealed that all three extracts of Q. velutina, C. procera and N. tabacum exhibited an effective antileishmanial activity and induced apoptosis against the L. tropica promastigotes. Further investigations are essential to isolate and analyze the chemical compositions and their biological properties.

Linguatulosis in small ruminants in southeastern Iran: Epidemiological, histopathological and phylogenetic findings and its public health importance.

Linguatulosis, as a zoonotic disease, can infect most ruminants and cause accidental infections in humans. The objective of this study was to explore the epidemiological, histopathological and phylogenetic profiles of Linguatula serrata infection in sheep and goats and its public health importance during 2015-2018. Mesenteric lymph nodes (MLNs) and liver tissue of goats and sheep were selected randomly in Kerman slaughterhouse. Nymphal samples were used for DNA extraction, amplification and subsequently phylogenetic analysis using 18s rRNA and cytochrome C oxidase subunit 1 (cox1). Overall, of 828 examined livestock, 179 (42.4%) goats and 71 (17.5%) sheep were found to be infected with the nymphal stage of L. serrata. A significant difference was observed between linguatulosis and age. In the histopathological assessment, longitudinal and transverse sections of L. serrata nymphs were observed within the cyst-like spaces surrounded by a wall of fine fibrosis and compact lymphocytes. Moreover, comparing with the L. serrata reference sequences, we found only a single nucleotide change in our goat haplotype in 18s genetic region; while much nucleotide variations were observed in cox1 gene sequences. The results of the present study showed a high infection rate among goats and sheep in southeastern Iran. A better understanding of the disease could be achieved when the parasite species, their molecular characterization and the extent of infection in the area are determined. It is fundamental to select a comprehensive control program in order to take proper preventive and therapeutic measures against the infection.

The potential role of nicotinamide on Leishmania tropica: An assessment of inhibitory effect, cytokines gene expression and arginase profiling.

Leishmaniasis represents a major health concern worldwide which has no effective treatment modality. Nicotinamide (NAm) has been used for a wide range of applications from anticancer to antimicrobial usage. This study aimed to assess the effect of NAm combination on Leishmania tropica Inhibition, as well as on cytokines gene expression and arginase (ARG) activity in L. tropica-infected macrophages in an in vitro model. The leishmanicidal effects of NAm and Glucantime (meglumine antimoniate, MA) alone and in combination (NAm/MA) were evaluated using a colorimetric assay and macrophage model. Additionally, immunomodulatory effects and enzymatic activity were assessed by analyzing Th1 and Th2 cytokines gene expression and ARG level, respectively, in infected macrophages treated with NAm and MA, alone and in combination. Findings indicated that the NAm/MA combination demonstrated greater inhibitory effects on L. tropica promastigotes and amastigotes compared with each drug individually. Docking results proved the affinity of NAm to IFN-γ, which can affirm the increased levels of IFN-γ, IL-12p40 and TNF-α as well as reductions in IL-10 secretion with a dose-response effect, especially in the combination group. The NAm/MA combination also showed a significant reduction in the level of ARG activity at all concentrations used compared to each drug individually. These findings indicate higher effectiveness of NAm plus MA in reducing parasite growth, promoting immune response and inhibiting ARG level. This combination should be considered as a potential therapeutic regimen for treatment of volunteer patients with anthroponotic cutaneous leishmaniasis (ACL) in future control programs.

Differential expression of TLRs 2, 4, 9, iNOS and TNF-α and arginase activity in peripheral blood monocytes from glucantime unresponsive and responsive patients with anthroponotic cutaneous leishmaniasis caused by Leishmania tropica.

BACKGROUND: Detection of the mechanism of host/parasite interactions in unresponsive forms of anthroponotic cutaneous leishmaniasis (ACL) caused by Leishmania tropica is helpful for immunotherapy and vaccine development. In the present study, the gene expression of toll-like receptors (TLRs), TNF-α, iNOS and also arginase (ARG) activity in monocytes from Glucantime unresponsive in comparison to responsive patients infected with L. tropica was investigated. METHODS: In this case-control study, patients with unresponsive (n = 10) and responsive (n = 10) ACL were recruited. Gene expression of TLR2, TLR4, TLR9, TNF-α and iNOS was analyzed in L. tropica-exposed monocytes. The level of ARG activity in both isolated promastigotes and the lysates of monocytes was also determined. RESULTS: L. tropica-exposed monocytes represented higher expression of all three TLRs and TNF-α and lower expression of iNOS compared to unexposed ones in both groups of patients. Results revealed a significant down-regulation of TLR2 and TNF-α and up-regulation of TLR9 expression in unresponsive isolates in comparison to responsive ones. Besides, ARG level showed a significant increase in L. tropica-stimulated monocytes and cultured promastigotes from unresponsive isolates versus responsive ones. CONCLUSIONS: The decreased TLR2, TLR4, TNF-α and iNOS and the increased level of TLR9 expression in L. tropica-exposed monocytes from unresponsive isolates and also the increment in ARG activity in their promastigotes and monocytes, might possibly be involved in the severity of the disease and leading to Glucantime unresponsiveness.

A single-group trial of end-stage patients with anthroponotic cutaneous leishmaniasis: Levamisole in combination with Glucantime in field and laboratory models.

Currently, there is no satisfactory treatment modality available for cutaneous leishmaniasis (CL). The major objective of the present study was to explore the effect of immunomodulator-levamisole in combination with Glucantime in end-stage unresponsive patients with anthroponotic CL (ACL). Twenty end-stage unresponsive patients with ACL were identified for participation in this single-group trial study. Simultaneously, each patient was received a combination of levamisole pills along with Glucantime during the remedy course. Several in vitro complementary experiments were performed to evaluate the mode of action of levamisole and Glucantime alone and in combination using a macrophage model, in vitro MTT assay, flow cytometry and quantitative real time PCR (qPCR). Overall, 75% of the patients showed complete clinical cure, 10% partially improved and the remaining (15%) had underlying chronic diseases demonstrated no response to the treatment regimen. In in vitro studies, there was no cytotoxic effect associated with these drugs in the range of our experiments. The findings by the flow cytometric analysis represented that the highest apoptotic values corresponded to the drugs combination (32.23%) at 200 µg/ml concentration. Finally, the gene expression level of IL-12 p40, iNOS and TNF-α promoted while the level of IL-10 and TGF-ß genes reduced as anticipated. The findings clearly indicated that the combination of levamisole and Glucantime should be considered in end-stage unresponsive patients with ACL who have not responded to basic treatments. The immunomodulatory role of levamisole in mounting immune system as documented by the in vitro experiments and further substantiated by this single-group trail study was highlighted.

In vitro Protoscolicidal Effects of Cinnamomum zeylanicum Essential Oil and Its Toxicity in Mice.

BACKGROUND: This study investigates the scolicidal effects of Cinnamomum zeylanicum essential oil against the protoscoleces of hydatid cysts and its toxicity in the mice model. MATERIALS AND METHODS: Gas chromatography/mass spectroscopy analyses were used to identify the constituents of essential oil. Protoscoleces were treated with different concentrations of the essential oil (6.25-100 µL/mL) in each test tube for 5-30 min. The viability of protoscoleces was confirmed using eosin exclusion test (0.1% eosin staining). Forty-eight male NMRI mice were also used to determine the toxicity of C. zeylanicum essential oil (0.5-4 mL/kg). RESULTS: The main components were found to be cinnamaldehyde (91.8%), ρ metoxicinamate (1.57%), and α pinene (1.25%). Findings indicate that C. zeylanicum essential oil with the concentrations of 100 and 50 µL/mL killed 100% of protoscoleces after 5 min of exposure. Also, the lower concentrations of C. zeylanicum essential oil motivated a late protoscolicidal effect. The LD50 value of intraperitoneal injection of C. zeylanicum essential oil was 2.07 mL/kg body weight after 48 h, and the maximum nonfatal dose was 1.52 mL/kg body weight. The results also showed that there was no significant toxicity following oral administration of C. zeylanicum essential oil for 2 weeks. CONCLUSION: The results exhibited the favorable scolicidal activity of C. zeylanicum, which could be applied as a natural scolicidal agent in hydatid cyst surgery. SUMMARY: We evaluated the efficacy of Cinnamomum zeylanicum essential oil against hydatid cyst protoscolecesThe viability of protoscoleces was confirmed using eosin exclusion test (0.1% eosin staining)Forty-eight male NMRI mice were also used to determine the toxicity of C. zeylanicum essential oilC. zeylanicum with potent scolicidal activity could be applied as a natural scolicidal agent in surgery. Abbreviations used: GC/MS: Gas chromatography/mass spectrometry analysis; CE: Cystic echinococcosis; LD50: Lethal dose 50%; I.p: Intraperitoneally.