Vol 27 No 2 December - The Indian Society for Parasitology

JPD : Vol. 27 (2), 2003
CSF production induced by L. donovani amastigote components
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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
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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,
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(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
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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;
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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