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in Pulmonary Host Defense in Murine Invasive Aspergillosis1
Department of Medicine, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical School, Ann Arbor, MI 48109
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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in other
pulmonary infections, we hypothesized that TNF- is an important
proximal signal in murine invasive pulmonary aspergillosis.
Intratracheal challenge with Aspergillus fumigatus
conidia in both neutropenic (cyclophosphamide-treated) and
nonneutropenic BALB/c mice resulted in the time-dependent increase in
lung TNF- levels, which correlated with the histologic development
of a patchy, peribronchial infiltration of mononuclear and
polymorphonuclear cells. Ab-mediated neutralization of TNF- resulted
in an increase in mortality in both normal and cyclophosphamide-treated
animals, which was associated with increased lung fungal burden as
determined by histology and as quantified by chitin content. Depletion
of TNF- resulted in a reduced lung neutrophil influx in both normal
and cyclophosphamide-treated animals, which occurred in association
with a decrease in lung levels of the C-X-C chemokine, macrophage
inflammatory protein-2 and the C-C chemokines macrophage inflammatory
protein-1 and JE. In cyclophosphamide-treated animals, intratracheal
administration of a TNF- agonist peptide (TNF70–80) 3
days before, but not concomitant with, the administration of
Aspergillus conidia resulted in improved survival from
9% in control mice to 55% in TNF70–80-treated animals.
These studies indicate that TNF- is a critical component of innate
immunity in both immunocompromised and immunocompetent hosts, and that
pretreatment with a TNF- agonist peptide in a compartmentalized
fashion can significantly enhance resistance to A.
fumigatus in neutropenic animals. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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The respiratory tract is the portal of entry in the majority of cases
of human invasive aspergillosis (3). It is postulated that innate
immunity is the principal pathway by which A. fumigatus is
cleared from the lung. This innate response consists of alveolar
macrophages, which represent the first line of defense against conidia
entering the alveolus, and recruited neutrophils. Neutrophils are
believed to kill conidia that have survived to form hyphae and cause
invasive infection (5). Various cells of macrophages lineage can engulf
and kill Aspergillus conidia in vitro (6, 7, 8) and in vivo (9, 10). Both macrophages (6, 11) and polymorphonuclear cells (5, 12) have
been shown to damage hyphae in vitro. When exposed to conidia in vitro,
cells of macrophage lineage secrete inflammatory mediators, including
the proximal cytokines TNF- and IL-1 (13, 14), as well as cytokines
that promote Th-1 phenotype immune responses (15).
TNF- is a 17-kDa cytokine secreted predominantly by various
macrophage populations, including alveolar macrophages. It has shown to
be a critical proximal signal in the initiation and maintenance of
innate pulmonary immunity in animal models of pneumonia (16, 17) and
human pneumonia (18). In the single study examining the role of TNF-
in invasive aspergillosis in vivo, immunocompetent animals administered
A. fumigatus conidia i.v. had improved survival when treated
with systemic murine rTNF- (19). The role of TNF- in pulmonary
host defense against A. fumigatus has not been defined.
Given that TNF- has been shown to be a critical mediator of innate
immunity against several respiratory pathogens, attempts have been made
to administer TNF- to augment innate and acquired host responses.
However, the therapeutic administration of TNF- is limited by
substantial dose-related toxicity, particularly when this cytokine is
administered systemically. A TNF- agonist peptide composed of the 11
amino acids that constitute the binding site of native human
TNF- to its receptors (referred to as
TNF70–80)3 has
recently been characterized (20, 21). Binding of TNF70–80
to TNF- receptors (both p55 and p75) has been shown to mediate many
leukocyte-activating effects of native TNF-, including stimulation
of neutrophils for enhanced protease release and respiratory burst, and
enhancement of neutrophil phagocytic activity and killing. When
administered systemically, TNF70–80 was associated with
less toxicity than that observed with native TNF-, due in part to
the fact that this peptide did not alter adhesive properties of the
endothelium (20). This peptide has been successfully administered
directly into the lungs of mice with Gram-negative bacterial pneumonia,
although the beneficial effects of TNF70–80 on bacterial
clearance and survival were only observed when the peptide was
administered 3 or 7 days before, but not concomitant with, bacterial
challenge (22).
In the current study we hypothesized that TNF- is an important
proximal signal in murine invasive pulmonary aspergillosis. We tested
this hypothesis by assessing the production of TNF- in the lungs of
normal and immunocompromised mice challenged with A.
fumigatus conidia. We next examined the effect of TNF-
depletion on survival, the burden of fungal organisms, and the host
inflammatory response. Finally, the effects of intrapulmonary delivery
of native TNF- or TNF70–80 on survival in
cyclophosphamide-treated animals were determined.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Polyclonal anti-murine TNF-, MIP-1, JE, MIP-2, and
KC Abs used in the ELISAs were produced by immunization of
rabbits with murine recombinant cytokines in multiple intradermal sites
with CFA. Carrier-free murine rTNF, MIP-1, JE, MIP-2, and KC were
purchased from R&D Systems (Minneapolis, MN). In TNF- neutralization
experiments, 0.5 ml of rabbit anti-murine TNF- serum or control
rabbit serum was administered i.p. 1 day before A.
fumigatus administration. The i.p. injections of 0.25 ml of
antiserum or control rabbit serum were repeated every 48 h for two
additional doses. TNF70–80 and control peptides were
synthesized at the University of Michigan Protein and Carbohydrate
Structure Facility and were composed of the amino acid sequences
H-Pro-Ser-Thr-His-Val-Leu-Ileu-Thr-His-Thr-Ileu-OH and
H-Gly-Gly-Asp-Pro-Gly-Ileu-Val-Thr-His-Ser-OH, respectively (20). Both
peptides were purified by HPLC and mass spectrometry, and contained no
detectable LPS, as determined by Limulus lysate assay
(ICN Biomedicals, Costa Mesa, CA).
Animals
Specific pathogen-free BALB/c mice (8-wk-old females; Charles River Breeding Laboratories, Wilmington, MA) were used in all experiments. All mice were housed in specific pathogen-free conditions within the animal care facility at the University of Michigan until the day of sacrifice. Immunocompromised animals were treated with 200 mg/kg of cyclophosphamide (Sigma, St. Louis, MO), administered i.p., 4 days before intratracheal (i.t.) inoculation. Treatment with cyclophosphamide resulted in peripheral blood neutropenia (absolute neutrophil count, <200 cells/µl) by days 4 and 6 after treatment, with a return of peripheral counts to pretreatment levels by day 10.
Preparation and administration of A. fumigatus conidia
We chose to use A. fumigatus strain 13073 (American Type Culture Collection, Manassas, VA) in our studies, as this strain has previously been shown to induce invasive aspergillosis in immunocompromised mice (23). The organism was grown on Sabouraud dextrose agar plates (Becton Dickinson, Cockeysville, MD) for 7–10 days at 37°C. The surface of each plate was then washed with 100 ml of sterile 0.1% Tween-80 (SigmaUltra, St. Louis, MO) in normal saline. The resulting suspension of conidia was filtered through sterile gauze to remove clumps and hyphal debris, and then washed once and resuspended in 4 ml of 0.1% Tween-80. The concentration of Aspergillus conidia in the suspension was determined by a particle counter (Z2 particle analyzer, Coulter, Hialeah, FL). The suspension was then diluted to the desired concentration, and the concentration was again measured before administration. In preliminary experiments, the number of particles determined by the particle counter was in close agreement with the number of viable colony-forming units found by serial dilution and plating of the suspension. On the day of inoculation, each animal was anesthetized with 1.8–2 mg of pentobarbital i.p. Using standard aseptic technique, the trachea was exposed and a 30-µl inoculum (A. fumigatus suspension or 0.1% Tween-80) was administered via a sterile 26-gauge needle. The skin incision was closed with surgical staples. In all experiments, immunocompetent animals were challenged with 1–2 x 107 conidia, and cyclophosphamide-treated animals were challenged with 1–2 x 106 conidia.
Lung harvest
At designated time points, the mice were sacrificed by CO2 asphyxiation. The chest cavity was opened aseptically, and the pulmonary vasculature was perfused with PBS via the right ventricle. For histologic examination, lungs were perfused with 1 ml of 4% paraformaldehyde in PBS, inflated with 1 ml of 4% paraformaldehyde in PBS via the trachea, and then excised en bloc. Lungs for various assays were perfused with 1 ml of PBS containing 5 mM EDTA, removed, frozen in liquid nitrogen, and stored at -20°C until the day of the assay. Lungs for cytokine and myeloperoxidase (MPO) assays were homogenized in 1 ml of 2x complete protease inhibitor mixture buffer (Boehringer Mannheim, Mannheim, Germany) in PBS using a tissue homogenizer (Biospec Products, Bartlesville, OK). A 900-µl aliquot of PBS was added to 900 µl from each sample, sonicated for 10 s, and centrifuged at 500 x g for 10 min. Supernatants were passed through a 0.45-µm pore size filter (Gelman, Ann Arbor, MI) and stored at 4°C for cytokine ELISA.
Lung chitin assay
Given that molds (including the Aspergillus species) do not reliably form reproductive units in tissue, we employed an assay for chitin to measure the burden of organisms in lungs. Chitin, a component of the hyphal wall, is absent from mammalian cells and conidia. The assay was adapted from a previously described method (24). Lungs were homogenized in 5 ml of distilled water and centrifuged (1500 x g, 5 min, 20°C). The supernatants were discarded, and pellets were resuspended in sodium lauryl sulfate (3%, w/v) and heated at 100°C for 15 min. Samples were then centrifuged (1500 x g, 5 min, 20°C), and pellets were washed with distilled water and resuspended in 3 ml of KOH (120%, w/v). Samples were heated at 130°C for 60 min. After cooling, 8 ml of ice-cold ethanol (75%, v/v) was added to each sample, and tubes were shaken until ethanol and KOH made one phase. Samples were incubated on ice for 15 min, and 0.3 ml of Celite suspension (supernatant of 1 g of Celite 545 (Fisher Scientific, Pittsburgh, PA) added to 75% ethanol and allowed to stand for 2 min) was added to each. Samples were centrifuged (1500 x g, 5 min, 4°C), and supernatants were discarded. Pellets were washed once with ethanol (40%, v/v) and twice with distilled water, and resuspended in 0.5 ml of distilled water. Standards, consisting of 0.2 ml of distilled water and 0.2 ml of glucosamine (10 µg/ml), were made up. NaNO2 (0.2 ml; 5%, w/v) and KHSO4 (0.2 ml; 5%, w/v) were added to each standard, and NaNO2 (0.5 ml; 5%, w/v) and KHSO4 (0.5 ml; 5%, w/v) were added to each tissue prep; all samples were mixed gently for 15 min and then centrifuged (1500 x g, 2 min, 4°C). Two 0.6-ml aliquots of supernatant from each tissue prep were transferred to separate tubes. Ammonium sulfamate (0.2 ml) was added to each tube, and all tubes were shaken vigorously for 5 min. A fresh solution of 3-methyl-2-thiazolone hydrazone HCl monohydrate (50 mg in 10 ml of distilled water) was made, and 0.2 ml was added to each tube. Samples were then heated to 100°C for 3 min and cooled. FeCl3·6H2O (0.2 ml; 0.83%, w/v) was added to each, and OD was measured at 650 nm after 25 min. Chitin content, measured in glucosamine equivalents, was measured by the following formula: chitin content = [5 x (OD of organ - OD of control organ)]/(OD of glucosamine - OD of water).
Lung MPO activity
Lung MPO activity was measured as a marker of neutrophil sequestration, as described previously (25). Briefly, a 100-µl aliquot of each lung homogenate was added to 100 µl of a buffer containing 50 mM potassium phosphate (pH 6.0), 5% hexadecyltrimthylammonium bromide, and 5 mM EDTA. Samples were sonicated for 10 s and centrifuged at 3000 xg for 15 min. The supernatant was mixed 1/15 with assay buffer and read at 490 nm. MPO units were calculated as the change in absorbance over time.
Cytokine ELISA
Murine TNF-, MIP-1, JE, MIP-2, and KC were quantified
using a modification of a double ligand method, as described previously
(26). Briefly, flat-bottom 96-well microtiter plates (Immuno-Plate I
96-F, Nunc, Copenhagen, Denmark) were coated with 50 µl/well of
rabbit anti-cytokine Ab (1 µg/ml in 0.6 M NaCl, 0.26 M
H3BO4, and 0.08 N NaOH, pH 9.6) for 16 h
at 4°C and then washed with PBS (pH 7.5) and 0.05% Tween-20 (wash
buffer). Microtiter plate nonspecific binding sites were blocked with
2% BSA in PBS and incubated for 90 min at 37°C. Plates were rinsed
four times with wash buffer, and undiluted and diluted (1/10) cell-free
supernatants (50 µl) in duplicate were added followed by incubation
for 1 h at 37°C. Plates were washed four times, followed by the
addition of 50 µl/well biotinylated rabbit anti-cytokine Abs (3.5
µg/ml in PBS (pH 7.5), 0.05% Tween-20, and 2% FCS), and plates were
incubated for 30 min at 37°C. Plates were washed four times,
streptavidin-peroxidase conjugate (Bio-Rad, Richmond, CA) was added,
and plates were incubated for 30 min at 37°C. Plates were washed
again four times, and chromogen substrate (Bio-Rad, Richmond, CA) was
added. The plates were incubated at room temperature to the desired
extinction, and the reaction was terminated with 50 µl/well of 3 M
H2SO4 solution. Plates were read at 490 nm in
an ELISA reader. Standards were 1/2 log dilutions of murine rTNF,
MIP-1, JE, MIP-2, or KC, from 1 pg/ml to 100 ng/ml. This ELISA
method consistently detected the relevant cytokine at concentrations
>25 pg/ml. The ELISAs did not cross-react with IL-1, IL-2, IL-4, IL-6,
IL-10, or IFN-
. In addition, each ELISA did not cross-react with
other members of the murine chemokine family.
Statistical analysis
Data were analyzed by a Power Macintosh 8600/300 computer using the InStat version 2.01 statistical package (GraphPad, San Diego, CA). Survival data were compared using the Fisher’s exact test. All other data were expressed as the mean ± SEM and compared using unpaired two-tailed Mann-Whitney (nonparametric) test. The p values were considered statistically significant if they were <0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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To characterize the host response to Aspergillus in
immunocompetent and immunocompromised animals, normal and
cyclophosphamide-treated BALB/c mice were challenged i.t. with
1–2 x 107 and 1–2 x 106 A.
fumigatus conidia, respectively. Lungs were examined
histologically at various time points after challenge. By 2 days after
A. fumigatus administration in both normal and
cyclophosphamide-treated mice, infiltration of inflammatory cells was
noted within the alveolar and interstitial compartments in a patchy,
peribronchial pattern (Fig. 1
). The
inflammatory cell infiltrate consisted predominantly of neutrophils
and, to a lesser extent, mononuclear cells. By 4 days after A.
fumigatus administration, cellular infiltrates were primarily
mononuclear, with resolution occurring in immunocompetent mice, whereas
a continued inflammatory response was noted in cyclophosphamide-treated
animals up to 7 days after challenge (data not shown). Conidia were
visible in normal mice 2 days after fungal inoculation and were nearly
completely cleared by 5 days. In contrast, hyphae (the invasive form of
A. fumigatus) were present only in cyclophosphamide-treated
mice and were most numerous by 2–3 days after A. fumigatus
administration. The inflammatory infiltrates observed corresponded to
areas of conidial deposition in normal mice and to hyphae in
cyclophosphamide-treated mice.
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expression after administration of A.
fumigatus conidia
We next correlated changes in lung TNF- levels with the
development of pulmonary inflammation in normal and
cyclophosphamide-treated mice after administration of A.
fumigatus conidia. As shown in Fig. 2A, a rapid increase in lung
TNF- levels was noted in immunocompetent mice when they were
challenged with 1–2 x 107 conidia, which reached a
plateau by 24 h. Administration of 1–2 x
106 A. fumigatus conidia to
cyclophosphamide-treated mice resulted in a marked elevation in lung
TNF- levels by 1 day after administration, with maximal TNF-
levels at 2 days, and a return to baseline levels by 4 days after
A. fumigatus administration (Fig. 2B). No
significant induction of TNF- was observed in the lungs of
immunocompetent animals challenged with 1–2 x
106 A. fumigatus conidia or in
cyclophosphamide-treated animals challenged with 0.1% Tween-80
(vehicle) i.t.
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neutralization on survival in normal and
immunocompromised mice inoculated with A. fumigatus
conidia
Additional studies were undertaken to evaluate the contribution of
TNF- to survival after inoculation of conidia. To ascertain the
effectiveness of the polyclonal anti-murine TNF- Ab in depleting
TNF- in vivo, we measured lung TNF- levels in normal and
cyclophosphamide-treated animals passively immunized with
anti-TNF- Ab or control serum 1 day before inoculation with
A. fumigatus conidia. On the basis of earlier studies
showing time of maximum expression of TNF, lungs were harvested 1 day
after inoculation in normal mice and 2 days after inoculation in
cyclophosphamide-treated mice. Lung homogenate TNF- levels in normal
mice were 3.53 ± 0.54 in the control group and 0.13 ± 0.051
in the anti-TNF- group (mean ± SEM; n = 6
mice/group; p < 0.01). In cyclophosphamide-treated
animals, levels were 6.16 ± 0.70 in the control group and
0.080 ± 0.007 in anti-TNF- group (mean ± SEM;
n = 6 mice/group; p < 0.01).
Having demonstrated significant in vivo neutralization of TNF, normal
and cyclophosphamide-treated animals were treated with anti-murine
TNF- or control rabbit serum 1 day before challenge with conidia.
Normal and cyclophosphamide-treated mice were then inoculated with
1.0 x 107 and 9.3 x 105 conidia,
respectively. As shown in Fig. 3,
treatment with anti-murine TNF- Ab resulted in 14% mortality in
normal mice and 47% mortality in cyclophosphamide-treated mice,
compared with no deaths in normal and cyclophosphamide-treated mice
receiving control serum. All animals that survived beyond 7 days were
considered long term survivors (>4 wk; data not shown).
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neutralization on lung fungal burden in normal
and cyclophosphamide-treated mice
To determine the mechanism of increased lethality in mice
passively immunized with anti-TNF- Ab, we examined fungal burden
in lungs of infected animals by histology and assessment of lung chitin
content. Immunocompetent BALB/c mice or mice treated with
cyclophosphamide were given either anti-TNF- Ab or rabbit serum
1 day before challenge with 1–2 x 107
(immunocompetent) or 1–2 x 106
(cyclophosphamide-treated) A. fumigatus conidia. Animals
were then sacrificed after 3 days for histology and lung chitin
measurement. In immunocompetent mice, treatment with anti-TNF-
Ab resulted in the appearance of both conidial and hyphal forms at 3
days after the administration of conidia, whereas only conidial forms
of A. fumigatus were seen in animals receiving preimmune
serum (data not shown). In cyclophosphamide-treated animals, a marked
increase in the number of hyphae was noted in animal treated with
anti-TNF- Ab, as evident on histology, compared with that in
control animals (Fig. 4, B and
D). To quantitate differences in fungal burden, lung chitin
levels were determined in cyclophosphamide-treated animals. Lungs of
TNF-depleted animals had a greater than twofold increase in chitin
content compared with those of control animals (Fig. 5).
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neutralization on recruitment of inflammatory
cells
To determine whether impaired fungal clearance in TNF-depleted
animals occurred as a result of changes in lung neutrophil influx,
lungs from cyclophosphamide-treated and normal mice given
anti-TNF- Ab or control serum were harvested for estimation of
MPO activity, a surrogate measure of neutrophil presence. On the basis
of studies showing the time of maximum expression of MPO in normal and
cyclophosphamide-treated animals (not shown), lung MPO levels were
evaluated 1 day after inoculation in normal animals and 2 days after
inoculation in cyclophosphamide-treated animals. In normal animals
treated with anti-TNF, there was a 79% decrease in lung MPO levels
compared with control values at 1 day, indicating reduced neutrophil
recruitment in the absence of TNF. In cyclophosphamide-treated mice,
the lung MPO level was reduced by 45% in TNF-depleted animals compared
with that in controls at 2 days (Fig. 6).
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neutralization on expression of C-X-C and C-C
chemokines
Given that TNF- has been shown to be a potent inducer of
both C-X-C and C-C chemokines, levels of the C-X-C chemokines MIP-2 and
KC, and of the C-C chemokines, MIP-1 and JE, were measured in lung
homogenates from normal and cyclophosphamide-treated mice. On the basis
of preliminary studies showing the time of maximum expression of these
chemokines, levels were measured 1 day after Aspergillus
inoculation in normal mice, and 3 days after Aspergillus
inoculation in cyclophosphamide-treated mice. In both normal and
cyclophosphamide-treated animals, there was a significant decrease in
MIP-2 levels in anti-TNF-treated animals challenged with conidia
compared with that in control animals receiving serum. No significant
change in KC levels was detected in either group. Treatment of normal
or cyclophosphamide-treated animals with anti-TNF- serum also
resulted in significant reduction in the C-C chemokines MIP-1 and
JE/monocyte chemoattractant protein-1 (Fig. 7 and Fig. 8).
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TNF70–80 on survival in invasive pulmonary aspergillosis
Having demonstrated that TNF- depletion increases fungal burden
and mortality, we next examined the effect of compartmentalized TNF-
augmentation on survival in cyclophosphamide-treated animals challenged
with A. fumigatus. To avoid systemic effects, we
administered either rmTNF- (1 µg/animal) or TNF70–80
(10 µg/animal) i.t. concomitant with the administration of A.
fumigatus conidia (1.8 x 106). Neither rmTNF-
nor TNF70–80 administration altered survival in
cyclophosphamide-treated animals when given at the time of
Aspergillus challenge compared with that in animals
receiving a control peptide of vehicle control (data not shown).
However, significantly enhanced survival was observed in
cyclophosphamide-treated mice that were pretreated with
TNF70–80 i.t. 3 days before the administration of A.
fumigatus compared with that in animals pretreated with either
control peptide or vehicle 3 days before inoculation with A.
fumigatus conidia (Fig. 9).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We studied the pulmonary host response to A. fumigatus in normal and immunocompromised animal models. Despite the 10-fold greater concentration of conidia used in all experiments involving immunocompetent animals, normal hosts readily cleared these large inocula without developing invasive disease. Cyclophosphamide, an alkylating agent in common clinical use as a chemotherapeutic and immunosuppressive agent, was used to render animals susceptible to invasive disease. Cyclophosphamide induces a variety of effects on immunologically active cells; these include a dose-dependent depletion of circulating neutrophils as well as alterations in the number and function of circulating and pulmonary monocyte/macrophages and various lymphocyte populations (27, 28). Although peripheral blood neutropenia is an easily measurable clinical risk factor for the development of invasive aspergillosis in humans, this susceptibility may also be related to the numerous other effects of cyclophosphamide on other immune effector cells. In our model, animals pretreated with cyclophosphamide before inoculation with Aspergillus conidia developed an acute, severe pneumonia that clinically and histologically resembled invasive pulmonary aspergillosis complicating severe immunosuppression in humans. Therefore, this model has the advantage of notable clinical relevance, but is complicated by the diverse effects of cyclophosphamide on various immune effector cells.
In this study we demonstrated that in both normal and
cyclophosphamide-treated mice, TNF- is produced in the lungs in
response to the i.t. administration of A. fumigatus. In
immunocompetent animals, the TNF- level is significantly elevated by
8 h after A. fumigatus inoculation. Resident alveolar
macrophages are most likely to be the source of this early and
localized release of TNF, which occurs before significant recruitment
of other immune effector cells occurs (data not shown). This view is
supported by the fact that TNF- is released from alveolar
macrophages coincubated with A. fumigatus conidia in vitro
(13). Elevated TNF- at later time points probably represents release
from various recruited cells, including neutrophils, and recruited
monocyte/macrophages. Notably, neutrophils were present in the lungs of
cyclophosphamide-treated mice infected with A. fumigatus
while the animals had peripheral blood neutropenia. This is presumably
due to preferential accumulation of neutrophils at the site of
inflammation despite a reduced pool of available neutrophils.
In vivo depletion of TNF- resulted in increased fungal burden and
mortality, which occurred in association with a reduction in lung
neutrophil infiltration. TNF- is likely to contribute to host
defense against A. fumigatus by several mechanisms. Although
not directly chemotactic for neutrophils, TNF- induces leukocyte and
endothelial cell expression of adhesion molecules, thus influencing
neutrophil trafficking in the lungs (29). In addition, our studies
indicate that the neutralization of TNF- resulted in significant
reductions in both C-X-C and especially C-C chemokines. Previous
studies have suggested a role for chemokines in host defense against
A. fumigatus: isolated rat alveolar macrophages produce
MIP-1, MIP-2, and KC, as well as TNF- in response to A.
fumigatus conidia in vitro (30), and knockout mice lacking CCR1, a
receptor for MIP-1 and RANTES, develop disseminated infection when
administered A. fumigatus i.v. (31). In this context, our
study provides evidence that TNF- drives the expression of both
C-X-C and C-C chemokines, including MIP-1, JE, and MIP-2, which may
in part mediate the influx and activation of neutrophils and
mononuclear cells in the lungs. Reduced amounts of MIP-2 and MIP-1
may explain the reduction in early neutrophil influx (and possibly
activation), increased fungal burden, and increased mortality in
TNF-depleted animals. In addition, MIP-1 and JE/monocyte
chemoattractant protein-1 may play important roles in mononuclear cell
recruitment and activation in the setting of invasive pulmonary
aspergillosis. Studies establishing causal relationships are in
progress.
The intratracheal administration of TNF70–80 provided
survival benefits when given 3 days before the i.t. inoculation of
A. fumigatus conidia, but not when given concomitantly with
conidia. These results parallel our earlier observations in murine
bacterial pneumonia (22). The lack of a beneficial effect in animals
treated with TNF70–80 concomitant with conidial
administration may be due to several possible mechanisms. Firstly,
TNF70–80 is more potent as a priming agent than as a
direct activator of leukocyte respiratory burst and degranulation in
vitro (20). Therefore, pretreatment with TNF70–80 may
provide sufficient macrophage priming, whereas concomitant treatment
may result in insufficient leukocyte activation. Alternatively,
leukocyte release of proteolytic enzymes in response to infectious
organisms within the airspace may lead to cytokine degradation and loss
of biologic effects. Lastly, the administration of native TNF- or
TNF70–80 given at the time of fungal challenge may induce
lung injury in the setting of pneumonia, thereby negating any
beneficial effect on mortality.
Given that immunocompromised patients at risk of developing pulmonary invasive aspergillosis are easily identifiable, early institution of prophylactic immunomodulatory treatment is a potential application of this study. Such clinical applications require further study.
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Acknowledgments |
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serum, and to Mrs. Mary Glass for technical assistance
in performing ELISAs. |
Footnotes |
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2 Address correspondence and reprint requests to Dr. Theodore J. Standiford, Division of Pulmonary and Critical Care Medicine, University of Michigan Medical Center, 6301 MSRB III, 1150 West Medical Center Drive, Ann Arbor, MI 48109-0360. E-mail address:
3 Abbreviations used in this paper: TNF70–80, TNF- agonist peptide; MIP-1; macrophage inflammatory protein-1; MIP-2, macrophage inflammatory protein-2; i.t., intratracheal; MPO, myeloperoxidase; CCR-1, C-C chemokine receptor-1.
Received for publication July 6, 1998. Accepted for publication October 8, 1998.
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  A. J. Hartigan, J. Westwick, G. Jarai, and C. M. Hogaboam CCR7 Deficiency on Dendritic Cells Enhances Fungal Clearance in a Murine Model of Pulmonary Invasive Aspergillosis J. Immunol., October 15, 2009; 183(8): 5171 - 5179. [Abstract] [Full Text] [PDF]
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 S. J. Park and B. Mehrad Innate Immunity to Aspergillus Species Clin. Microbiol. Rev., October 1, 2009; 22(4): 535 - 551. [Abstract] [Full Text] [PDF]
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T. R. T. Dagenais and N. P. Keller Pathogenesis of Aspergillus fumigatus in Invasive Aspergillosis Clin. Microbiol. Rev., July 1, 2009; 22(3): 447 - 465. [Abstract] [Full Text] [PDF]
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J. L. Werner, A. E. Metz, D. Horn, T. R. Schoeb, M. M. Hewitt, L. M. Schwiebert, I. Faro-Trindade, G. D. Brown, and C. Steele Requisite Role for the Dectin-1 {beta}-Glucan Receptor in Pulmonary Defense against Aspergillus fumigatus J. Immunol., April 15, 2009; 182(8): 4938 - 4946. [Abstract] [Full Text] [PDF]
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S. J. Park, M. A. Hughes, M. Burdick, R. M. Strieter, and B. Mehrad Early NK Cell-Derived IFN-{gamma} Is Essential to Host Defense in Neutropenic Invasive Aspergillosis J. Immunol., April 1, 2009; 182(7): 4306 - 4312. [Abstract] [Full Text] [PDF]
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 A.-P. Bellanger, L. Millon, K. Khoufache, D. Rivollet, I. Bieche, I. Laurendeau, M. Vidaud, F. Botterel, and S. Bretagne Aspergillus fumigatus germ tube growth and not conidia ingestion induces expression of inflammatory mediator genes in the human lung epithelial cell line A549 J. Med. Microbiol., February 1, 2009; 58(2): 174 - 179. [Abstract] [Full Text] [PDF]
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 M Manz, C Beglinger, and S R Vavricka Fatal invasive pulmonary aspergillosis associated with adalimumab therapy Gut, January 1, 2009; 58(1): 149 - 149. [Full Text] [PDF]
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 H. Ramaprakash, T. Ito, T. J. Standiford, S. L. Kunkel, and C. M. Hogaboam Toll-Like Receptor 9 Modulates Immune Responses to Aspergillus fumigatus Conidia in Immunodeficient and Allergic Mice Infect. Immun., January 1, 2009; 77(1): 108 - 119. [Abstract] [Full Text] [PDF]
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 M. Simitsopoulou, E. Roilides, F. Paliogianni, C. Likartsis, J. Ioannidis, K. Kanellou, and T. J. Walsh Immunomodulatory Effects of Voriconazole on Monocytes Challenged with Aspergillus fumigatus: Differential Role of Toll-Like Receptors Antimicrob. Agents Chemother., September 1, 2008; 52(9): 3301 - 3306. [Abstract] [Full Text] [PDF]
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E. J. Cornish, B. J. Hurtgen, K. McInnerney, N. L. Burritt, R. M. Taylor, J. N. Jarvis, S. Y. Wang, and J. B. Burritt Reduced Nicotinamide Adenine Dinucleotide Phosphate Oxidase-Independent Resistance to Aspergillus fumigatus in Alveolar Macrophages J. Immunol., May 15, 2008; 180(10): 6854 - 6867. [Abstract] [Full Text] [PDF]
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 J. R. Slusher, M. E. Maldonado, F. Mousavi, and C. J. Lozada Central nervous system Aspergillus fumigatus presenting as cranial nerve palsy in a patient with ankylosing spondylitis on anti-TNF therapy Rheumatology, May 1, 2008; 47(5): 739 - 740. [Full Text] [PDF]
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C. Bretz, G. Gersuk, S. Knoblaugh, N. Chaudhary, J. Randolph-Habecker, R. C. Hackman, J. Staab, and K. A. Marr MyD88 Signaling Contributes to Early Pulmonary Responses to Aspergillus fumigatus Infect. Immun., March 1, 2008; 76(3): 952 - 958. [Abstract] [Full Text] [PDF]
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 S. Tsiodras, G. Samonis, D. T. Boumpas, and D. P. Kontoyiannis Fungal Infections Complicating Tumor Necrosis Factor {alpha} Blockade Therapy Mayo Clin. Proc., February 1, 2008; 83(2): 181 - 194. [Abstract] [Full Text] [PDF]
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T. E. Rodriguez, N. R. Falkowski, J. R. Harkema, and G. B. Huffnagle Role of Neutrophils in Preventing and Resolving Acute Fungal Sinusitis Infect. Immun., December 1, 2007; 75(12): 5663 - 5668. [Abstract] [Full Text] [PDF]
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 T. M. Hohl and M. Feldmesser Aspergillus fumigatus: Principles of Pathogenesis and Host Defense Eukaryot. Cell, November 1, 2007; 6(11): 1953 - 1963. [Full Text] [PDF]
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 B. H. Segal Role of Macrophages in Host Defense Against Aspergillosis and Strategies for Immune Augmentation Oncologist, October 1, 2007; 12(suppl_2): 7 - 13. [Abstract] [Full Text] [PDF]
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 A. P. Phadke, G. Akangire, S. J. Park, S. A. Lira, and B. Mehrad The Role of CC Chemokine Receptor 6 in Host Defense in a Model of Invasive Pulmonary Aspergillosis Am. J. Respir. Crit. Care Med., June 1, 2007; 175(11): 1165 - 1172. [Abstract] [Full Text] [PDF]
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M. Simitsopoulou, E. Roilides, C. Likartsis, J. Ioannidis, A. Orfanou, F. Paliogianni, and T. J. Walsh Expression of Immunomodulatory Genes in Human Monocytes Induced by Voriconazole in the Presence of Aspergillus fumigatus Antimicrob. Agents Chemother., March 1, 2007; 51(3): 1048 - 1054. [Abstract] [Full Text] [PDF]
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F. Schobel, O. Ibrahim-Granet, P. Ave, J.-P. Latge, A. A. Brakhage, and M. Brock Aspergillus fumigatus Does Not Require Fatty Acid Metabolism via Isocitrate Lyase for Development of Invasive Aspergillosis Infect. Immun., March 1, 2007; 75(3): 1237 - 1244. [Abstract] [Full Text] [PDF]
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M. Dubourdeau, R. Athman, V. Balloy, M. Huerre, M. Chignard, D. J. Philpott, J.-P. Latge, and O. Ibrahim-Granet Aspergillus fumigatus Induces Innate Immune Responses in Alveolar Macrophages through the MAPK Pathway Independently of TLR2 and TLR4 J. Immunol., September 15, 2006; 177(6): 3994 - 4001. [Abstract] [Full Text] [PDF]
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M. Lekkala, A. M. LeVine, M. J. Linke, E. C. Crouch, B. Linders, E. Brummer, and D. A. Stevens Effect of Lung Surfactant Collectins on Bronchoalveolar Macrophage Interaction with Blastomyces dermatitidis: Inhibition of Tumor Necrosis Factor Alpha Production by Surfactant Protein D. Infect. Immun., August 1, 2006; 74(8): 4549 - 4556. [Abstract] [Full Text] [PDF]
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G. M. Gersuk, D. M. Underhill, L. Zhu, and K. A. Marr Dectin-1 and TLRs Permit Macrophages to Distinguish between Different Aspergillus fumigatus Cellular States J. Immunol., March 15, 2006; 176(6): 3717 - 3724. [Abstract] [Full Text] [PDF]
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V. Gafa, R. Lande, M. C. Gagliardi, M. Severa, E. Giacomini, M. E. Remoli, R. Nisini, C. Ramoni, P. Di Francesco, D. Aldebert, et al. Human Dendritic Cells following Aspergillus fumigatus Infection Express the CCR7 Receptor and a Differential Pattern of Interleukin-12 (IL-12), IL-23, and IL-27 Cytokines, Which Lead to a Th1 Response Infect. Immun., March 1, 2006; 74(3): 1480 - 1489. [Abstract] [Full Text] [PDF]
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 I. Shalit, D. Halperin, D. Haite, A. Levitov, J. Romano, N. Osherov, and I. Fabian Anti-inflammatory effects of moxifloxacin on IL-8, IL-1{beta} and TNF-{alpha} secretion and NF{kappa}B and MAP-kinase activation in human monocytes stimulated with Aspergillus fumigatus J. Antimicrob. Chemother., February 1, 2006; 57(2): 230 - 235. [Abstract] [Full Text] [PDF]
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 N. Berkova, S. Lair-Fulleringer, F. Femenia, D. Huet, M.-C. Wagner, K. Gorna, F. Tournier, O. Ibrahim-Granet, J. Guillot, R. Chermette, et al. Aspergillus fumigatus conidia inhibit tumour necrosis factor- or staurosporine-induced apoptosis in epithelial cells Int. Immunol., January 1, 2006; 18(1): 139 - 150. [Abstract] [Full Text] [PDF]
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 C. D. Hamilton Immunosuppression Related to Collagen-Vascular Disease or Its Treatment Proceedings of the ATS, December 1, 2005; 2(5): 456 - 460. [Abstract] [Full Text] [PDF]
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A. Rivera, H. L. Van Epps, T. M. Hohl, G. Rizzuto, and E. G. Pamer Distinct CD4+-T-Cell Responses to Live and Heat-Inactivated Aspergillus fumigatus Conidia Infect. Immun., November 1, 2005; 73(11): 7170 - 7179. [Abstract] [Full Text] [PDF]
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K. J. Carpenter and C. M. Hogaboam Immunosuppressive Effects of CCL17 on Pulmonary Antifungal Responses during Pulmonary Invasive Aspergillosis Infect. Immun., November 1, 2005; 73(11): 7198 - 7207. [Abstract] [Full Text] [PDF]
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V. Balloy, M. Si-Tahar, O. Takeuchi, B. Philippe, M.-A. Nahori, M. Tanguy, M. Huerre, S. Akira, J.-P. Latge, and M. Chignard Involvement of Toll-Like Receptor 2 in Experimental Invasive Pulmonary Aspergillosis Infect. Immun., September 1, 2005; 73(9): 5420 - 5425. [Abstract] [Full Text] [PDF]
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 C. F. Benjamim, S. K. Lundy, N. W. Lukacs, C. M. Hogaboam, and S. L. Kunkel Reversal of long-term sepsis-induced immunosuppression by dendritic cells Blood, May 1, 2005; 105(9): 3588 - 3595. [Abstract] [Full Text] [PDF]
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N. Singh and D. L. Paterson Aspergillus Infections in Transplant Recipients Clin. Microbiol. Rev., January 1, 2005; 18(1): 44 - 69. [Abstract] [Full Text] [PDF]
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A. C. Herring, N. R. Falkowski, G.-H. Chen, R. A. McDonald, G. B. Toews, and G. B. Huffnagle Transient Neutralization of Tumor Necrosis Factor Alpha Can Produce a Chronic Fungal Infection in an Immunocompetent Host: Potential Role of Immature Dendritic Cells Infect. Immun., January 1, 2005; 73(1): 39 - 49. [Abstract] [Full Text] [PDF]
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S. D. Stephens-Romero, A. J. Mednick, and M. Feldmesser The Pathogenesis of Fatal Outcome in Murine Pulmonary Aspergillosis Depends on the Neutrophil Depletion Strategy Infect. Immun., January 1, 2005; 73(1): 114 - 125. [Abstract] [Full Text] [PDF]
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V. Balloy, M. Huerre, J.-P. Latge, and M. Chignard Differences in Patterns of Infection and Inflammation for Corticosteroid Treatment and Chemotherapy in Experimental Invasive Pulmonary Aspergillosis Infect. Immun., January 1, 2005; 73(1): 494 - 503. [Abstract] [Full Text] [PDF]
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 C. M. Hogaboam, K. Takahashi, R. A. B. Ezekowitz, S. L. Kunkel, and J. M. Schuh Mannose-binding lectin deficiency alters the development of fungal asthma: effects on airway response, inflammation, and cytokine profile J. Leukoc. Biol., May 1, 2004; 75(5): 805 - 814. [Abstract] [Full Text] [PDF]
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 C. F. Benjamim, C. M. Hogaboam, N. W. Lukacs, and S. L. Kunkel Septic Mice Are Susceptible to Pulmonary Aspergillosis Am. J. Pathol., December 1, 2003; 163(6): 2605 - 2617. [Abstract] [Full Text]
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M. J. Becker, S. de Marie, M. H. A. M. Fens, H. A. Verbrugh, and I. A. J. M. Bakker-Woudenberg Effect of amphotericin B treatment on kinetics of cytokines and parameters of fungal load in neutropenic rats with invasive pulmonary aspergillosis J. Antimicrob. Chemother., September 1, 2003; 52(3): 428 - 434. [Abstract] [Full Text] [PDF]
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H. L. Van Epps, M. Feldmesser, and E. G. Pamer Voriconazole Inhibits Fungal Growth without Impairing Antigen Presentation or T-Cell Activation Antimicrob. Agents Chemother., June 1, 2003; 47(6): 1818 - 1823. [Abstract] [Full Text] [PDF]
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 G. D. Brown, J. Herre, D. L. Williams, J. A. Willment, A. S. J. Marshall, and S. Gordon Dectin-1 Mediates the Biological Effects of {beta}-Glucans J. Exp. Med., May 5, 2003; 197(9): 1119 - 1124. [Abstract] [Full Text] [PDF]
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H. Hebart, C. Bollinger, P. Fisch, J. Sarfati, C. Meisner, M. Baur, J. Loeffler, M. Monod, J.-P. Latge, and H. Einsele Analysis of T-cell responses to Aspergillus fumigatus antigens in healthy individuals and patients with hematologic malignancies Blood, December 15, 2002; 100(13): 4521 - 4528. [Abstract] [Full Text] [PDF]
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B. Mehrad, M. Wiekowski, B. E. Morrison, S.-C. Chen, E. C. Coronel, D. J. Manfra, and S. A. Lira Transient Lung-Specific Expression of the Chemokine KC Improves Outcome in Invasive Aspergillosis Am. J. Respir. Crit. Care Med., November 1, 2002; 166(9): 1263 - 1268. [Abstract] [Full Text] [PDF]
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 S. S. Mambula, K. Sau, P. Henneke, D. T. Golenbock, and S. M. Levitz Toll-like Receptor (TLR) Signaling in Response to Aspergillus fumigatus J. Biol. Chem., October 11, 2002; 277(42): 39320 - 39326. [Abstract] [Full Text] [PDF]
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A. C. Herring, J. Lee, R. A. McDonald, G. B. Toews, and G. B. Huffnagle Induction of Interleukin-12 and Gamma Interferon Requires Tumor Necrosis Factor Alpha for Protective T1-Cell-Mediated Immunity to Pulmonary Cryptococcus neoformans Infection Infect. Immun., June 1, 2002; 70(6): 2959 - 2964. [Abstract] [Full Text] [PDF]
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 A. Warris, A. Bjorneklett, P. Gaustad, G. F. Keenan, T. F. Schaible, and J. A. Boscia Invasive Pulmonary Aspergillosis Associated with Infliximab Therapy N. Engl. J. Med., April 5, 2001; 344(14): 1099 - 1100. [Full Text] [PDF]
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J. K. Brieland, C. Jackson, F. Menzel, D. Loebenberg, A. Cacciapuoti, J. Halpern, S. Hurst, T. Muchamuel, R. Debets, R. Kastelein, et al. Cytokine Networking in Lungs of Immunocompetent Mice in Response to Inhaled Aspergillus fumigatus Infect. Immun., March 1, 2001; 69(3): 1554 - 1560. [Abstract] [Full Text] [PDF]
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B. Finkel-Jimenez, M. Wuthrich, T. Brandhorst, and B. S. Klein The WI-1 Adhesin Blocks Phagocyte TNF-{{alpha}} Production, Imparting Pathogenicity on Blastomyces dermatitidis J. Immunol., February 15, 2001; 166(4): 2665 - 2673. [Abstract] [Full Text] [PDF]
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K. Blease, B. Mehrad, N. W. Lukacs, S. L. Kunkel, T. J. Standiford, and C. M. Hogaboam Antifungal and Airway Remodeling Roles for Murine Monocyte Chemoattractant Protein-1/CCL2 During Pulmonary Exposure to Asperigillus fumigatus Conidia J. Immunol., February 1, 2001; 166(3): 1832 - 1842. [Abstract] [Full Text] [PDF]
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 F. Reichenberger, J.M. Habicht, A. Gratwohl, and M. Tamm Diagnosis and treatment of invasive pulmonary aspergillosis in neutropenic patients Eur. Respir. J., January 1, 2001; 19(4): 743 - 755. [Abstract] [Full Text] [PDF]
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H. Briscoe, D. R. Roach, N. Meadows, D. Rathjen, and W. J. Britton A novel tumor necrosis factor (TNF) mimetic peptide prevents recrudescence of Mycobacterium bovis bacillus Calmette-Guerin (BCG) infection in CD4+ T cell-depleted mice J. Leukoc. Biol., October 1, 2000; 68(4): 538 - 544. [Abstract] [Full Text]
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K. Blease, B. Mehrad, T. J. Standiford, N. W. Lukacs, J. Gosling, L. Boring, I. F. Charo, S. L. Kunkel, and C. M. Hogaboam Enhanced Pulmonary Allergic Responses to Aspergillus in CCR2-/- Mice J. Immunol., September 1, 2000; 165(5): 2603 - 2611. [Abstract] [Full Text] [PDF]
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K. Blease, B. Mehrad, T. J. Standiford, N. W. Lukacs, S. L. Kunkel, S. W. Chensue, B. Lu, C. J. Gerard, and C. M. Hogaboam Airway Remodeling Is Absent in CCR1-/- Mice During Chronic Fungal Allergic Airway Disease J. Immunol., August 1, 2000; 165(3): 1564 - 1572. [Abstract] [Full Text] [PDF]
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B. Mehrad, T. A. Moore, and T. J. Standiford Macrophage Inflammatory Protein-1{alpha} Is a Critical Mediator of Host Defense Against Invasive Pulmonary Aspergillosis in Neutropenic Hosts J. Immunol., July 15, 2000; 165(2): 962 - 968. [Abstract] [Full Text] [PDF]
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E. Cenci, A. Mencacci, A. Bacci, F. Bistoni, V. P. Kurup, and L. Romani T Cell Vaccination in Mice with Invasive Pulmonary Aspergillosis J. Immunol., July 1, 2000; 165(1): 381 - 388. [Abstract] [Full Text] [PDF]
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C. M. Hogaboam, K. Blease, B. Mehrad, M. L. Steinhauser, T. J. Standiford, S. L. Kunkel, and N. W. Lukacs Chronic Airway Hyperreactivity, Goblet Cell Hyperplasia, and Peribronchial Fibrosis during Allergic Airway Disease Induced by Aspergillus fumigatus Am. J. Pathol., February 1, 2000; 156(2): 723 - 732. [Abstract] [Full Text] [PDF]
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B. Mehrad, R. M. Strieter, T. A. Moore, W. C. Tsai, S. A. Lira, and T. J. Standiford CXC Chemokine Receptor-2 Ligands Are Necessary Components of Neutrophil-Mediated Host Defense in Invasive Pulmonary Aspergillosis J. Immunol., December 1, 1999; 163(11): 6086 - 6094. [Abstract] [Full Text] [PDF]
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