502 Wheat et al.
Leishmania major
, CD40 ligation played only a minor, if
blood. Plates were incubated for 10 days at 30oC, and
colony counts were determined.
any, role in recovery from histoplasmosis [9].
Here, we further examine the role of CD40–CD40L in
recovery from histoplasmosis using a murine model of
intratracheally induced infection.
Cytokine measurement
Upon sacrice, aliquots of lung and spleen homogenates
o
¡
were stored at 70 C for cytokine determination. IL-10,
Methods
g
IL-12 and IFN- concentrations were measured by
enzyme immunoassay (BioSource International Inc).
Immunodepletion of CD4 T lymphocytes
g
Homogenates were used undiluted for IL-12 and IFN-
Antibodies used for CD4 depletion were produced from
cell lines purchased from American Type Culture
Collection (ATCC, Manassas, VA, USA) and were
administered intraperitoneally as claried ascites uid
[10 every 7 days at a concentration that maintained CD4
cells below 1% for at least two weeks. Clone GK1.5
(ATCC TIB-207), rat anti-mouse L3T4 was used to
deplete mice of CD4 lymphocytes.
determination, whereas lung homogenates were diluted
1 : 5 and spleen homogenates were diluted 1 : 2 for IL-10
determinations.
Statistical analysis
A one-way analysis of variance (ANOVA) was per-
formed on the ranks of data within and across the
different groups. Pair wise comparison of all groups with
controls was done using Dunn’s all pairwise multiple
comparison procedure. A signicance level of 0.05 was
employed to test all hypotheses.
Preparation of H. capsulatum yeasts
H. capsulatum
The yeast phase of a clinical isolate of
,
IU-CT, was grown in HMM media [11] in a 37oC
incubator with shaking at 150 r.p.m. for 48 h. The yeast
culture was centrifuged and washed with Hank’s
‡
Results
Effect of CD40L on fungal burden in CD4-depleted
mice
balanced salt solution
20 mM HEPES. The inoculum
4
£
was adjusted using a hemocytometer to 1 10 yeasts in
m
25 l.
CD40L, beginning two days prior to infection infection,
lowered fungal burden in the lungs (median cfu g¡1
4.4 10 ), compared with untreated controls (8.8 10 ),
5 0.05 (Fig. 1). CD40L beginning two days after
infection also reduced fungal burden in the lungs
(6.2 10 ), as did low-dose amphotericin B (4.5 10 ),
5 0.05. CD40L did not enhance the effect of low-dose
amphotericin B, however, and none of these interven-
tions reduced the fungal burden in the spleen, 4 0.05
5
5
Mouse inoculation
£
£
P
Six-week-old B6C3F1 mice (Harlan Sprague–Dawley)
were anesthetized with 4.5% halothane at an oxygen
ow rate of 0.9 l m¡1. A 20 gauge, 1 1/4 inch angiocath
(Becton Dickinson) was passed to the bifurcation of the
trachea and the inoculum was injected, as described
previously [12].
5
5
£
£
P
P
for all.
CD40L and amphotericin B treatment
Effect of CD40L on fungal burden using a fresh isolate
of H. capsulatum in CD4-depleted mice
CD40L ¡w1 as given intraperitoneally at
a
dose of
‡
m
¡
35 g ml
beginning on either day 2 or 2 post
The fungal burden in the untreated controls was lower
than had been observed using the same inoculum in a
prior study [12], suggesting that the inoculum was less
virulent. Low-dose amphotericin B (0.2 mg kg¡1 every
other day) failed to reduce fungal burden in the earlier
study [12] but reduced fungal burden in the lung by
0.5 log in this study. Accordingly, a fresh isolate of the
infecting strain was prepared from the original frozen
stock culture. The fresh isolate induced mortality and
produced fungal burden results as expected for this
strain. This new isolate was used to repeat the fungal
burden studies and explore the effect of CD40L on
mortality.
inoculation and continued until day 14 for experiments
in CD4-depleted animals and from day 2 to day 4 in
¡
‡
non-immunosuppressed animals. Amphotericin B w¡as1
m
given intraperitoneally at doses of 2.0 or 0.2 g ml
every other day from day 4 to day 14 of infection.
Quantitative culture of organ homogenates
One day after the last drug dose, lungs and spleen were
harvested as described elsewhere [12]. Organs were
weighed, then ground in Tenbroeck tissue grinders
containing 2.0 ml RPMI-1640 medium. Organ homoge-
nates were plated on BHI agar containing 10% sheep
ã