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  Background and Objectives: Leishmanin skin test (Montenegro test) is a best indicator for evaluation of delayed-type hypersensitivity reaction and cell-mediated immunity in leishmaniasis. A standard antigen is needed for this test. In this research, several batches of leishmanin antigen were produced under standard conditions, and their immunogenicity, specificity, sensitivity and potency were evaluated.

  Methods: In order to produce leishmanin, standard strain of Leishmania major (MRHO/IR/75/ER) was cultured in equal volume of�liquid medium of D-MEM and Tc Medium 199 in large scale. Parasites from stationary phase of growth were harvested and washed under strict standard conditions and used for preparation of leishmanin. Immunogenicity of prepared antigen was tested by skin testing in pre-immunized guinea pigs. Specificity of the reagent and abnormal sensitization were evaluated by skin testing in healthy individuals in non-endemic areas of Tehran and Tabriz. Sensitivity and potency of leishmanin reagent were evaluated by skin testing in recovered individuals from cutaneous leishmaniasis (CL) in endemic areas of rural and urban areas.

  Results: The findings indicated the productions of leishmanin are sterile and safe with high immunogenicity. Specificity of the products was shown to be higher than 99% with no abnormal sensitization to reagent. Sensitivity and potency of preparations were determined > 96% with mean induration between 15-18 mm in endemic areas of rural CL, and > 93% with mean reactivity of 12-14 mm in endemic areas of urban CL.

  Conclusion: The findings indicated that this product is safe and sterile with high immunogenicity, specificity, sensitivity and potency and has no abnormal sensitization. These products which are easily available inside the country could be used easily for skin testing and detection of delayed-type hypersensitivity response in leishmaniasis.

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Background & objectives: Pentavalent antimonials are the first-line drugs for treatment of leishmaniasis, which have multiple side effects such as drug toxicity. Moreover, parasite resistance to these drugs is rising around the world. Second-line drugs, including Amphotericin B and pantamidine have also side effects and expensive for patients. According to the cytotoxic effects of paraquat, this study was conducted to evaluate the effect of paraquat on Leishmania major promastigotes and HUVECs viability.
Methods: A number of 2.5×10⁶ of Leishmania major promastigotes were treated in each well of 96 well plates with different concentrations of paraquat. Cells were incubated for 48 hours in 24 °C. MTT test was performed for evaluating paraquat impact on promastigotes. The absorbance was measured using a microplate reader at 570 nm.  The trypan blue staining assay was performed to evaluate the number of viable Leishmania major promastigotes following paraquat treatment. Furthermore, the effect of paraquat concentrations on HUVECs viability was evaluated under the cell culture condition.
Results: The results of the MTT test showed that increasing concentrations of paraquat could significantly reduce the viability and the number of Leishmania major promastigotes in comparison to control group (p<0.05). In this study, the IC₅₀ for Leishmania major promastigotes was calculated as 272.46 µg/ml. Trypan blue results were in line with the finding of MTT assay. Moreover, we found that HUVECs were susceptible to paraquat (IC₅₀=188.99 µg/ml).
Conclusion: Paraquat has a strong inhibitory effect on Leishmania major promastigotes and human endothelial cells. Although more comprehensive studies on the effects of the topical use of paraquat on Leishmania major lesions in animal model and its side effects are necessary.