Vol 27 No 2 December - The Indian Society for Parasitology

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86 CSF production induced by L. donovani amastigote components JPD : Vol. 27 (2), 2003 concentrations, and their constitutive levels are very passage in golden hamsters (Mesocrisetus auratus), low; however, during infections, the concentrations of intracardially (i.c.). Full-blown infection (150-200 CSFs are rapidly elevated (Cheers et al., 1988). amastigotes/100 spleen cell nuclei) was observed after Functionally, CSFs augment several immune 30-40 days, which was assessed by the microscopic parameters which include adherence, chemotaxis, examination of Giemsa-stained spleen touch prints respiratory burst activity, cytotoxicity, and increased (Singal et al., 2003). synthesis of various cytokines like interferon, tumor Harvesting of amastigotes and preparation of necrosis factor-α (TNF-α) and IL-1 (Grabstein et al., LDAA: Amastigotes were isolated from the spleen 1986; Warren and Ralph, 1986; Heidenreich et al., homogenates of heavily-infected hamsters, and 1989; Metcalf, 1991). CSFs are known to boost host treated with rbc-lysis buffer (Sigma-Aldrich, USA). defence against infections (Freund and Kleine, 1992; The amastigotes were washed (x2) with and suspended Armitage, 1998; Buchsel et al., 2002; Hubel et al., in sterile Dulbecco's modified Eagle medium 2002). (DMEM; Gibco, USA). For LDAA, the amastigotes GM-CSF has been reported to activate MØs for the were freeze-thawed twice, sonicated for 30 s at 4°C killing of Leishmania parasites, in vitro (Handman and (x3), and then centrifuged at 3000 g for 20 min at 4°C. Burgess, 1979; Weiser et al., 1987; Ho et al., 1990). In The clear LDAA-containing supernatant (0.41 mg vivo treatment with recombinant mouse GM-CSF protein/ml) was harvested aseptically, filter-sterilized (rmGM-CSF) has beneficial effects, whereas (0.2 µ) and stored at -70°C. Control antigen was treatment with rabbit anti-mouse GM-CSF antibodies prepared similarly by using splenocytes from normal has detrimental effects on the course of L. donovani male hamsters. A single preparation of LDAA was infection in mice (Murray et al., 1995). Injection of used in these studies. BALB/c mice with GM-CSF-activated MØs Generation of rabbit anti-LDAA serum: LDAA previously incubated with L. major antigens has also (400 µg) in complete Freund's adjuvant (FA; 1:1) was been reported to protect against subsequent injected in a rabbit at four different subcutaneous (s.c.) homologous challenge (Doherty and Coffman, 1993). Increased hematopoiesis has been reported in murine sites, followed by a booster of 200 µg of LDAA in leishmaniasis, which is probably driven by CSFs incomplete FA, s.c., four weeks later. A second booster (Mirkovich et al., 1986; Lelchuk et al., 1988; Cotterell was given one week later, with 100 µg of LDAA, et al., 2000a) as evidenced by the enhanced expression intravenously (i.v.), and the rabbit was finally bled for of GM-CSF mRNA both in vitro (Cotterell et al., anti-LDAA serum, three days after the last boost. The 2000b) and in vivo (Murray et al., 1995). Herein, serum was heat-inactivated (HI; 56°C; 30 min) and apparently for the first time, we report the CSF- stored at -20°C. The serum had an antibody titre induction potential of L. donovani amastigote antigens 1:2048 as determined by enzyme-linked soluble in culture medium (LDAA), both in vivo and in immunosorbent assay. vitro. Macrophages: For peritoneal MØs, thioglycollateinjected MATERIALS AND METHODS (4% wt/vol; 0.5 ml/mouse; 96 h) normal mice peritoneal exudates cell suspension was centrifuged Animals and parasite: BALB/c mice (18-20 g; male) (700 g; 7 min; 4°C), and the cell pellet was were obtained from the Central Animal Facility of the 6 resuspended (1x10 cells/ml) in 10 ml DMEM institute, and maintained at 22-24°C with food and supplemented with 10% fetal bovine serum (FBS, water provided ad libitum. All studies were carried out Gibco), 2 mM L-glutamine (Gibco), 0.01 M HEPES in accordance with the guidelines for Care and Use of -4 (Gibco), 1x10 M 2-mercaptoethanol (2-ME, Sigma) Animals in Scientific Research, Indian National and 40 µg/ml gentamicin (Gibco; CDMEM), and cell Science Academy, New Delhi, as adapted and promulgated by the Institutional Animal Ethics culture-tested polymyxin B sulfate (5 µg/ml; Sigma). Committee. L. donovani (MHCM/IN/80/Dd8 strain) The splenic MØs were obtained from splenocyte was maintained by cryopreservation and serial suspensions made by using a 20 µ nylon sieve, and the cells were then washed by centrifugation (700 g; 12
JPD : Vol. 27 (2), 2003 CSF production induced by L. donovani amastigote components 87 min; 4°C). Erythrocytes were lysed with rbc-lysis mononuclear cells were obtained by density gradient buffer, and the cells were suspended in CDMEM. For centrifugation of normal mouse femur BM cells. The 6 bone marrow (BM)-derived MØs, mouse femurs were cells were washed (x3) and suspended (5xl0 cells/ml) flushed with chilled DMEM using a 24 G needle, and in CDMEM. The non-adherent cells were separated by washed (x2) and suspended in CDMEM. The adherent adherence/depletion processes, and resuspended MØs from these three cell suspensions were harvested, 4 (2xl0 cells/ml) in CDMEM without FBS but separately, by allowing them to attach with the plastic containing 30% HI-horse serum (Gibco), 0.9% surface of T-25 culture flasks at 37°C for 3 h in 5% methylcellulose (Sigma), 0.9% deionized bovine CO2-air atmosphere, and were then further incubated -4 serum albumin (Sigma) and 1xl0 M 2-ME. The test for 30 min in an equal volume of CDMEM containing 2 and control serum and CM samples were added to this µg/ml indomethacin (Sigma). The MØs were detached cell suspension at 5% and 10% concentrations, by using a sterile rubber scraper, washed (x3) and respectively. One ml cultures of this cell suspension resuspended in 5 ml chilled polymyxin B-free Hanks' were then established in sterile 35 mm plastic dishes balanced salt solution (HBSS, Gibco). T-cells from and incubated at 37°C in humid 5% CO2-air these MØs were eliminated by rabbit anti-mouse T-cell atmosphere for 14 days. Following incubation, the serum (1:20) treatment for 1 h at 4°C followed by number of colonies with 50 or more cells was counted HBSS wash (xl), and incubation with rabbit under a dark-field inverted microscope (40x). The complement (1 h; 37°C). The DMEM and HBSS colonies were fixed on the glass slides and stained with contained 96% pure as determined by morphologic, type of CSF produced, the serum and CM samples phagocytic and non-specific esterase staining criteria, were incubated (37°C; 30 min) with excess amounts of and were >98% viable as judged by Trypan Blue neutralizing concentrations of anti-mouse G-CSF-, M- exclusion. CSF- or GM-CSF-specific goat polyclonal IgG (R & D Systems, Inc., USA), and then assayed for the residual Generation of CSFs: A single injection of LDAA CSF activity. (0.01, 0.1, 1, 5 and 10 mg/kg) was administered in mice (i.v.; 6 mice/dose), and their blood samples were GM colony forming units (CFU-GM) assay: The collected aseptically, after 2, 6, 12, 24, 48 and 72 h. CFU-GM count in single cell suspensions prepared Sera from these blood samples were separated and from the spleens and BM of LDAA (0.01-10.0 mg/kg)- pooled for each time-point, separately and used to treated mice were determined in colony-forming estimate serum CSFs. For controls, pooled sera assays, performed in semi-solid cultures (Yap and obtained from mice injected with muramyl dipeptide Stevenson, 1992; Riopel et al., 2001). Briefly, spleen (MDP; 25 µg/kg), DMEM (vehicle) and HI-LDAA cells, obtained by mincing the spleens and passing (70°C; 1 h; pH 7.0) were used. In vitro, different through a sterile nylon mesh (20 µ), were suspended in concentrations of LDAA (0.01-1 mg/ml) were added 15 ml of DMEM containing 10% FBS, 2% HEPES and to the cultured peritoneal, splenic and BM-derived 40 µg/ml gentamicin and centrifuged (700 g; 12 min; 4 6 MØs (1x10 -1x10 cells/ml; 3 ml/dish) and their CM 4°C). Erythrocytes were lysed with rbc-lysis buffer, were collected aseptically after various time intervals the cells were washed with DMEM, and erythrocyte (2-72 h), centrifuged (1000 g; 10 min; 4°C) and filter- ghosts were removed by filtering cell suspensions sterilized (0.2 µ). For controls, CM of MØs treated through sterile gauze. BM cells were flushed out from with MDP (1 µg/ml), CDMEM only or HI-LDAA were the mice femurs with 1 ml of cold Iscove's modified used. All the sera and CM were stored at -20°C until Dulbecco's medium (IMDM; Gibco) supplemented use. with 5% FBS, 40 µg/ml gentamicin and 2 mM L- glutamine. The spleen and BM cell suspensions were Measurement of CSF activity: CSFs were measured washed (x3) and suspended at a concentration of 4 x in terms of their colony-stimulating activity (Nanno et 6 10 cells/ml in IMDM. The cells were >96% viable as al., 1988; Singh and Dutta, 1991). Briefly, determined by Trypan Blue exclusion. The CFU-GM
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Vol 27 No 2 December - The Indian Society for Parasitology

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