Parasitology Research

, Volume 112, Issue 4, pp 1427–1435 | Cite as

Leishmania donovani-specific 25- and 28-kDa urinary proteins activate macrophage effector functions, lymphocyte proliferation and Th1 cytokines production

  • Vinod Kumar
  • Jalaj K. Gour
  • Nisha Singh
  • Surabhi Bajpai
  • Rakesh K. Singh
Original Paper


Growing incidence of drug resistance against leishmaniasis in endemic areas and limited drug options necessitates the need for a vaccine. Notwithstanding significant leishmanial research in the past decades, a vaccine candidate is far from reality. In this study, we report the potential of two urinary leishmanial proteins to induce macrophage effector functions, inflammatory cytokines production and human lymphocytes proliferation. A total four proteins of molecular mass 25, 28, 54 and 60 kDa were identified in human urine samples. The 25 and 28 kDa proteins significantly induced NADPH oxidase (p < 0.001), superoxide dismutase (p < 0.001) and inducible nitric oxide synthase (p < 0.001) activities in stimulated RAW264.7 macrophages. The release of nitric oxide, tumor necrosis factor-alpha and interleukin (IL)-12 was also significantly (p < 0.001) higher in 25 and 28 kDa activated macrophages as compared with cells activated with other two proteins. These two proteins also induced significant (p < 0.001) proliferation and release of IFN-γ and IL-12 in human peripheral blood mononuclear cells.



Financial assistance received from Council of Scientific and Industrial Research (No. 27(0183)/08/EMR-II), New Delhi, India, and Department of Biotechnology (BT/PR11177/MED/29/2008), New Delhi, India, is greatly acknowledged. The authors VK, JKG, SB and NS are thankful to CSIR, UGC, CSIR and DBT, New Delhi, respectively, for their research fellowships.

Conflict of interest



  1. Alvar J, Vélez ID, Bern C, Herrero M, Desjeux P, Cano J, Jannin J, den Boer M (2012) Leishmaniasis worldwide and global estimates of its incidence. PLoS One 7(5):e35671PubMedCrossRefGoogle Scholar
  2. Assreuy J, Cunha FQ, Epperlein M, Noronha-Dutra A, O’Donnell CA, Liew FY, Moncada S (1994) Production of nitric oxide and superoxide by activated macrophages and killing of Leishmania major. Eur J Immunol 24:672–676PubMedCrossRefGoogle Scholar
  3. Bacellar O, D’oliveira A Jr, Jeronimo S, Carvalho EM (2002) IL-10 and IL-12 are the main regulatory cytokines in visceral leishmaniasis. Cytokine 12:1228–1231CrossRefGoogle Scholar
  4. Bharswaj S, Srivastava N, Sudan R, Saha B (2010) Leishmania interferes with host cell signaling to devise a survival strategy. J Biomed Biotechnol 2010: 109189 (online)Google Scholar
  5. Bhaumik SK, Naskar K, De T (2009) Complete protection against experimental visceral leishmaniasis with complete soluble antigen from attenuated Leishmania donovani promastigotes involves Th1-immunity and down-regulation of IL-10. Eur J Immunol 39:2146–2160PubMedCrossRefGoogle Scholar
  6. Castellano LR, Filho DC, Argiro L, Dessein H, Prata A, Dessein A, Rodrigues V (2009) Th1/Th2 immune responses are associated with active cutaneous leishmaniasis and clinical cure is associated with strong interferon-gamma production. Hum Immunol 70:383–390PubMedCrossRefGoogle Scholar
  7. Castellanos-Serra LR, Fernandez-Patron C, Hardy E, Santana H, Huerta V (1997) High yield elution of proteins from sodium dodecyl sulfate-polyacrylamide gels at the low picomole level. Application to N-terminal sequencing of a scarce protein and to in solution biological activity analysis of on-gel renatured proteins. J Protein Chem 16:415–419PubMedCrossRefGoogle Scholar
  8. Chakravarty J, Kumar S, Trivedi S, Rai VK, Singh A, Anshman JA, Laughlin EM, Coler RN, Kahn SJ, Beckmann AM, Cowgill KD, Reed SG, Sundar S, Piazza FM (2011) A clinical trial to evaluate the safety and immunogenicity of the LEISH-F1 + MPL-SE vaccine for use in the prevention of visceral leishmaniasis. Vaccine 29:3531–3537PubMedCrossRefGoogle Scholar
  9. Chakravarty J, Sundar S (2010) Drug resistance in leishmaniasis. J Glob Infect Dis 2:167–176PubMedCrossRefGoogle Scholar
  10. Ding AH, Nathan CF, Stuehr DJ (1988) Release of reactive nitrogen intermediates and reactive oxygen intermediates from mouse peritoneal macrophages: comparison of activating cytokines and evidence for independent production. J Immunol 141:2407–2412PubMedGoogle Scholar
  11. Engwerda CR, Ato M, Kaye PM (2004) Macrophages, pathology and parasite persistence in experimental visceral leishmaniasis. Trends Parasitol 20:524–530PubMedCrossRefGoogle Scholar
  12. Evans KJ, Kedzierski L (2012) Development of vaccine against leishmaniasis. J Trop Med 2012:892817PubMedGoogle Scholar
  13. Garg R, Gupta SK, Tripathi P, Hajela K, Sundar S, Naik S, Dube A (2006) Leishmania donovani: identification of stimulatory soluble antigenic proteins using cured human and hamster lymphocytes for their prophylactic potential against visceral leishmaniasis. Vaccine 24:2900–2909PubMedCrossRefGoogle Scholar
  14. Gautam S, Kumar R, Maurya R, Nylén S, Ansari N, Rai M, Sundar S, Sacks D (2011) IL-10 neutralization promotes parasite clearance in splenic aspirate cells from patients with visceral leishmaniasis. J Infect Dis 204:1134–1137PubMedCrossRefGoogle Scholar
  15. Guevara-Mendoza O, Une C, Franceschi Carreira P, Orn A (1997) Experimental infection of Balb/cmice with Leishmania panamensis and Leishmania mexicana induction of early IFN-γ but not IL-4 is associated with the development of cutaneous lesions. Scand J Immunol 46:35–40PubMedCrossRefGoogle Scholar
  16. Handman E, Bullen DV (2002) Interaction of Leishmania with the host macrophage. Trends Parasitol 18:332–334PubMedCrossRefGoogle Scholar
  17. Horta MF, Mendes BP, Roma EH, Noronha FS, Macêdo JP, Oliveira LS, Duarte MM, Vieira LQ (2012) Reactive oxygen species and nitric oxide in cutaneous leishmaniasis. J Parasitol Res. doi:10.1155/2012/203818
  18. Iles KE, Forman JA (2002) Macrophage signaling and respiratory burst. Immunol Res 26:95–105PubMedCrossRefGoogle Scholar
  19. Kumar V, Gour JK, Bajpai S, Mishra M, Singh RK (2011) Detection of urinary antigens and their seroreactivity with serum of patients in Leishmania donovani infection. Asian Pac J Trop Med 4:367–370PubMedCrossRefGoogle Scholar
  20. Kumar V, Mishra M, Rajput SK, Bajpai S, Singh RK (2012) Detection and diagnostic applicability of human urinary kininogen in kala-azar patients. Parasitol Res 111:1851–1855PubMedCrossRefGoogle Scholar
  21. Lowry OH, Rosebrough NJ, Farr AL, Randal RJ (1951) Protein measurement with folin- phenol reagent. J Biol Chem 193:265–275PubMedGoogle Scholar
  22. Mahapatra SK, Chakraborty SP, Roy S (2010) In-vitro time dependent nicotine- induced free radical generation and status of glutathione cycle in murine peritoneal macrophage. Al Ameen J Med Sci 3:182–194Google Scholar
  23. Miralles GD, Stoeckle MY, McDermott DF, Finkelman FD, Murray HW (1994) Th1 and Th2 cell-associated cytokines in experimental visceral leishmaniasis. Infect Immun 62:1058–1063PubMedGoogle Scholar
  24. Mishra BB, Singh RK, Srivastava A, Tripathi VJ, Tiwari VK (2009) Fighting against leishmaniasis: search of alkaloids as future true potential anti-leishmanial agents. Mini Rev Med Chem 9:107–123PubMedCrossRefGoogle Scholar
  25. Misra HP, Fridovich I (1972) The role of superoxide anion in the autoxidation of epinephrine and a simple assay for superoxide dismutase. J Biol Chem 247:3170–3175PubMedGoogle Scholar
  26. Mosmann TR, Coffman RL (1989) Th1 and Th2 cells: different patterns of lymphokine secretion lead to different functional properties. Annu Rev Immunol 7:145–173PubMedCrossRefGoogle Scholar
  27. Nagill R, Kaur S (2011) Vaccine candidates for leishmaniasis: a review. Int Immunopharmacol 11:1464–1488PubMedCrossRefGoogle Scholar
  28. Nylen S, Sacks D (2007) Interleukin-10 and the pathogenesis of human visceral leishmaniasis. Trends Immunol 28:378–384PubMedCrossRefGoogle Scholar
  29. Rhee SG, Kang SW, Jeong W, Chang TS, Yang KS, Woo HA (2005) Intracellular messenger function of hydrogen peroxide and its regulation by peroxiredoxins. Curr Opin Cell Biol 17:183–189PubMedCrossRefGoogle Scholar
  30. Romao S, Castro H, Sousa C, Carvlho S, Tomas AM (2009) The cytosolic tryparedoxin of Leishmania infantum is essential for parasite survival. Int J Parasitol 39:703–711PubMedCrossRefGoogle Scholar
  31. Sacks DL, Lal SL, Shrivastava SN, Blackwell J, Neva FA (1987) An analysis of T cell responsiveness in Indian kala-azar. J Immunol 138:908–913PubMedGoogle Scholar
  32. Saha S, Mondal S, Banerjee A, Ghose J, Bhowmick S, Ali N (2006) Immune responses in kala-azar. Indian J Med Res 123:245–266PubMedGoogle Scholar
  33. Sharma U, Singh S (2009) Immunobiology of leishmaniasis. Indian J Exp Biol 47:412–423PubMedGoogle Scholar
  34. Singh N, Kumar M, Singh RK (2012) Leishmaniasis: current status of available drugs and new potential drug targets. Asian Pac J Trop Med 5(485):497Google Scholar
  35. Singh RK, Pandey HP, Sundar S (2006) Visceral leishmaniasis (kala-azar): challenges ahead. Indian J Med Res 123:331–344PubMedGoogle Scholar
  36. Srivastava A, Singh N, Mishra M, Kumar V, Gour JK, Bajpai S, Singh S, Pandey HP, Singh RK (2012) Identification of toll-like receptor related Th1 responsive Leishmania donovani amastigote specific antigens. Mol Cell Biochem 359:359–368PubMedCrossRefGoogle Scholar
  37. Stanley AC, Engwerda CR (2007) Balancing immunity and pathology in visceral leishmaniasis. Immunol Cell Biol 85:138–147PubMedCrossRefGoogle Scholar
  38. Trachootham D, Lu W, Ogaswara MA, Nilsa RD, Huang P (2008) Redox regulation of cell survival. Antioxid Redox Signal 10:1343–1374PubMedCrossRefGoogle Scholar
  39. Winberg ME, Rasmusson B, Sundqvist T (2007) Leishmania donovani: inhibition of phagosomal maturation is rescued by nitric oxide in macrophages. Exp Parasitol 117:165–170PubMedCrossRefGoogle Scholar
  40. Winyard PG, Moody CJ, Jacob C (2005) Oxidative activation of antioxidant defence. Trends Biochem Sci 30:453–461PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Vinod Kumar
    • 1
  • Jalaj K. Gour
    • 1
  • Nisha Singh
    • 1
  • Surabhi Bajpai
    • 1
  • Rakesh K. Singh
    • 1
  1. 1.Molecular Immunology Laboratory, Department of Biochemistry, Faculty of ScienceBanaras Hindu UniversityVaranasiIndia

Personalised recommendations