In vivo/In vitro immune responses to L. major isolates from patients with no clinical response to Glucantime


1 Skin and Stem Cell Research Center, Tehran University of Medical Sciences, Tehran; Department of Parasitology and Mycology, Isfahan University of Medical Sciences, Isfahan, Iran

2 Medical Parasitology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran

3 Center for Research and Training in Skin Diseases and Leprosy, Tehran University of Medical Sciences, Tehran, Iran

4 Bio-Statistics and Epidemiology, School of Health, Isfahan University of Medical Sciences, Isfahan, Iran

5 Biology and Genetics, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran

6 Pharmaceutics, Novel Drug Delivery Systems Research Center, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran

7 Clinical Biochemistry Department, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan, Iran

8 Department of Parasitology and Mycology, School of Medicine, Skin Diseases and Leishmaniasis Research Center, Isfahan University of Medical Sciences, Isfahan; Medical Parasitology, School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran


Background: Leishmaniasis is a major health problem in some endemic areas of tropical and subtropical areas of the world. Interleukin-12 (IL-12) and interferon gamma (IFN-γ) are essential cytokines associated with initiation of Th1 response. The main objective of this study was to evaluate of the type of immune response to L. major isolates from patients with no clinical response to antimonite (Glucantime).
Materials and Methods: This experimental study was carried out during 2013–2014. In the current study Leishmania major were isolated from 10 CL patients with a history of at least one course of treatment with Meglumine antimonate (Sb5). The isolates were used to evaluatein vitro andin vivo response to Sb5. J774 murine macrophage cell line was used forin vitro tests and Balb/c mice was used forin vivo studies. IL-12 gene expression was evaluated using Real-time PCR and IFN-γ serum level was quantified using ELISA technique. SPSS (version: 20), analysis of Covariance (ANCOVA) was used for statistical analysis.
Results: PCR results confirmed that all 10 isolates were L. major. The mean of IL-12 gene expression in vitro,in vivo and IFN-γ serum levels (pg/ml) after 2 and 3 weeks treatment in vivo, increased significantly following the treatment with Glucantime in the two groups of Balb/c mice infected either with patients' isolates or standard L. major. No significant difference was seen between the patients' isolates and standard species.
Conclusions: Although the L. major were isolated from patients with active lesion and no clinical response to Glucantime after at least one courses of Glucantime treatment butin vivo andin vitro immune response of L. major isolates showed no difference between the patients' isolates and standard L. major.


Organization WH. Control of the leishmaniases: Report of a meeting of the WHO Expert Commitee on the Control of Leishmaniases. Geneva: WHO Technical Report Series, 2010.  Back to cited text no. 1
Bogdan C. Leishmaniasis in rheumatology, haematology and oncology: Epidemiological, immunological and clinical aspects and caveats. Ann Rheum Dis 2012;71(Suppl 2):i60-6.  Back to cited text no. 2
Peters NC, Egen JG, Secundino N, Debrabant A, Kimblin N, Kamhawi S, et al.In vivo imaging reveals an essential role for neutrophils in leishmaniasis transmitted by sand flies. Science 2008;321:970-4.  Back to cited text no. 3
Sharma U, Singh S. Immunobiology of leishmaniasis. Indian J Exp Biol 2009;47:412-23.  Back to cited text no. 4
Khamesipour A. Therapeutic vaccines for leishmaniasis. Expert Opin Biol Ther 2014;14:1-9.  Back to cited text no. 5
Rostami MN, Keshavarz H, Edalat R, Sarrafnejad A, Shahrestani T, Mahboudi F, et al. CD8+T cells as a source of IFN-γ production in human cutaneous leishmaniasis. PLoS Negl Trop Dis 2010;4:845.  Back to cited text no. 6
O'Daly JA, Spinetti HM, Gleason J, Rodríguez MB. Clinical and immunological analysis of cutaneous leishmaniasis before and after different treatments. J Parasitol Res 2013;2013:657016.  Back to cited text no. 7
Hamza T, Barnett JB, Li B. Interleukin 12 a key immunoregulatory cytokine in infection applications. Int J Mol Sci 2010;11:789-806.  Back to cited text no. 8
Satoskar AR, Rodig S, Telford III SR, Satoskar AA, Ghosh SK, von Lichtenberg F, et al. IL-12 gene-deficient C57BL/6 mice are susceptible to Leishmania donovani but have diminished hepatic immunopathology. Eur J Immunol 2000;30:834-9.  Back to cited text no. 9
Sacks D, Noben-Trauth N. The immunology of susceptibility and resistance to Leishmania major in mice. Nat Rev Immunol 2002;2:845-58.  Back to cited text no. 10
Swihart K, Fruth U, Messmer N, Hug K, Behin R, Huang S, et al. Mice from a genetically resistant background lacking the interferon gamma receptor are susceptible to infection with Leishmania major but mount a polarized T helper cell 1-type CD4+T cell response. New York: Rockefeller Univ Press; 1995. p. 961-71.  Back to cited text no. 11
Noyes HA, Reyburn H, Bailey JW, Smith D. A nested-PCR-based schizodeme method for identifying Leishmania kinetoplast minicircle classes directly from clinical samples and its application to the study of the epidemiology of Leishmania tropica in Pakistan. J Clin Microbiol 1998;36:2877-81.  Back to cited text no. 12
Esmaeili J, Mohebali M, Edrissian GH, Rezayat SM, Ghazi-Khansari M, Charehdar S. Evaluation of miltefosine against leishmania major (MRHO/IR/75/ER):In vitro andin vivo studies. Acta Medica Iranica 2008;46:191-6.  Back to cited text no. 13
Overbergh L, Valckx D, Waer M, Mathieu C. Quantification of murine cytokine mRNAs using real time quantitative reverse transcriptase PCR. Cytokine 1999;11:305-12.  Back to cited text no. 14
Ohkusu K, Yoshimoto T, Takeda K, Ogura T, Kashiwamura SI, Iwakura Y, et al. Potentiality of interleukin-18 as a useful reagent for treatment and prevention of Leishmania major infection. Infect Immun 2000;68:2449-56.  Back to cited text no. 15
Hosmani AH, Thorat Y, Kasture P. Carbopol and itspharmaceutical significance: A review. Latest Reviews 2006;4:1-13.  Back to cited text no. 16
Alvar J, Velez ID, Bern C, Herrero M, Desjeux P, Cano J, et al. Leishmaniasis worldwide and global estimates of its incidence. PLoS One 2012;7:e35671.  Back to cited text no. 17
World Health Organization. Available from: [Last accessed on 7 April 2014].  Back to cited text no. 18
Chawla B, Madhubala R. Drug targets in Leishmania. J Parasit Dis 2010;34:1-13.  Back to cited text no. 19
Croft SL, Olliaro P. Leishmaniasis chemotherapy-challenges and opportunities. Clin Microbiol Infect 2011;17:1478-83.  Back to cited text no. 20
Sundar S. Drug resistance in Indian visceral leishmaniasis. Trop Med Int Health 2001;6:849-54.  Back to cited text no. 21
Sundar S, Chakravarty J. Leishmaniasis: An update of current pharmacotherapy. Expert Opin Pharmacother 2013;14:53-63.  Back to cited text no. 22
Basu JM, Roy S. Sodium antimony gluconate (SAG) mediates antileishmanial effect by stimulating innate and cellular arms of the immune system. Antimicrob Agents Chemother 2006;50:1788-97.  Back to cited text no. 23
Croft SL, Yardley V. Chemotherapy of leishmaniasis. Curr Pharm Des 2002;8:319-42.  Back to cited text no. 24
Awasthi A, Mathur RK, Saha B. Immune response to Leishmania infection. Indian J Med Res 2004;119:238-58.  Back to cited text no. 25
Murray H, Oca M, Granger A, Schreiber R. Requirement for T cells and effect of lymphokines in successful chemotherapy for an intracellular infection. Experimental visceral leishmaniasis. J Clin Invest 1989;83:1253.  Back to cited text no. 26
Ghosh M, Roy K, Roy S. Immunomodulatory effects of antileishmanial drugs J Antimicrob Chemother 2013;68:2834-8.  Back to cited text no. 27
Munder M, Mallo M, Eichmann K, Modolell M. Murine macrophages secrete interferon γ upon combined stimulation with IL-12 and IL-18: A novel pathway of autocrine macrophage activation. J Exp Med 1998;187:2103-8.  Back to cited text no. 28
Schmitt E, Hoehn P, Huels C, Goedert S, Palm N, Rüde E, et al. T helper type 1 development of naive CD4+T cells requires the coordinate action of interleukin-12 and interferon-γ and is inhibited by transforming growth factor-β. Eur J Immunol 1994;24:793-8.  Back to cited text no. 29
Wadhone P, Maiti M, Agarwal R, Kamat V, Martin S, Saha B. Miltefosine promotes IFN-γ-dominated anti-leishmanial immune response. J Immunol 2009;182:7146-54.  Back to cited text no. 30
Murray HW, Delph-Etienne S. Roles of endogenous gamma interferon and macrophage microbicidal mechanisms in host response to chemotherapy in experimental visceral leishmaniasis. Infect Immun 2000;68:288-93.  Back to cited text no. 31
Asadpour A, Riazi-Rad F, Khaze V, Ajdary S, Alimohammadian M. Distinct strains of Leishmania major induce different cytokine mRNA expression in draining lymph node of BALB/c mice. Parasite Immunol 2013;35:42-50.  Back to cited text no. 32