Lung (2005) 183:239--251

DOI: 10.1007/s00408-004-2535-y

Gammaherpesvirus-Induced Lung Pathology Is

Altered in the Absence of Macrophages

J. M. Cadillac,1* R. E. Sigler,2 J. B. Weinberg,3 M. L. Lutzke,3** and

R. Rochford4

1
Unit for Laboratory Animal Medicine, University of Michigan, Ann Arbor, Michigan 48109, USA;
2
Esperion Therapeutics, Inc., 3621 S. State St., 695 KMS Place, Ann Arbor, Michigan 48108, USA;
3
Division of Pediatric Infectious Diseases, University of Michigan, Ann Arbor, Michigan 48109, USA;
4
Department of Microbiology and Immunology, SUNY Upstate Medical University,
750 East Adams St., Syracuse New York 13210, USA

Abstract. The purpose of this study was to examine the lung pathogenesis of
murine gammaherpesvirus (MHV-68) infection in mice that lack CC chemo-
kine receptor CCR2, an important receptor for macrophage recruitment to
sites of inflammation. BALB/c and CCR2)/) mice were inoculated intranasally
(i.n.) with MHV-68 and samples were collected during acute infection (6 dpi)
and following viral clearance (12 dpi). Immunohistochemistry was used to
determine which cells types responded to MHV-68 infection in the lungs. Lung
pathology in infected BALB/c mice was characterized by a mixed inflammatory
cell infiltrate, necrosis, and increased alveolar macrophages by 12 dpi.
Immunohistochemistry showed intense positive staining for macrophages.
CCR2)/) mice showed greater inflammation in the lungs at 12 dpi than did
BALB/c mice, with more necrosis and diffuse neutrophil infiltrates in the
alveoli. Immunohistochemistry demonstrated much less macrophage infiltra-
tion in the CCR2)/) mice than in the BALB/c mice. These studies show that
CCR2 is involved in macrophage recruitment in response to MHV-68 infection
and illustrates how impairments in macrophage function affect the normal
inflammatory response to this viral infection.

Key words: Macrophage—Lung—Gammaherpesvirinae/pathogenicity—Dis-

ease models, animal—Chemokines.

*Current address: Laboratory Animal Health Sciences, The Jackson Laboratory, 600 Main St.,
Bar Harbor, Maine
**Current address: Kent County Health Department, Grand Rapids, Michigan
Correspondence to: R. Rochford; email: rochforr@upstate.edu
240 J. M. Cadillac et al.

Introduction

The lung is often the site of entry for viruses because of the large bronchial and
alveolar surface area. Although many viral infections are rapidly resolved, evi-
dence is accumulating that is a subset of viral infections is associated with chronic
lung disease. For example, herpesvirus infections have been shown to be associ-
ated with the development of idiopathic pulmonary fibrosis [33] and primary
pulmonary hypertension [7]. Understanding how viral infections induce immu-
nopathology in the lungs is critical to development of prevention and treatment of
chronic lung diseases.
Challenge of mice with a variety of respiratory viruses has proven to be a
useful model system to elucidate many of the host factors involved in immune
response in the lung. Infection of mice with murine gammaherpesvirus (MHV)-68
serves as a model to study the effects of viral infection on lung pathology. MHV-68
is a naturally occurring rodent pathogen which, following intranasal (i.n.) inocu-
lation in mice, replicates in the lungs [1, 5, 22, 24, 27, 29, 31, 32]. Viral latency is
established predominantly in the mediastinal lymph nodes (MLN) and in the
spleen [5, 29], but there is also evidence that viral DNA can persist in the lungs of
infected mice [28, 37] suggesting that this virus could also contribute to a sustained
immune response in the lungs.
Chemokines are chemotactic cytokines that orchestrate the movement of
leukocytes along a concentration gradient into and out of sites of inflammation
[18, 25, 34]. They are increasingly recognized as key mediators of inflammation
and leukocyte recruitment in response to inflammatory stimuli including viral
infections [18, 25]. Viral respiratory infection induces the production of chemo-
kines critical to the host inflammatory response [9, 10, 13, 20, 36].
Early descriptions of lung inflammation following MHV-68 infection noted
cellular infiltrates lasting up to 30 days post inoculation (dpi) [2, 30]. We and
others have described a sustained chemokine response, including monocyte
chemoattractant protein-1 (MCP-1), to lung infection with MHV-68 [26, 38]
suggesting that these chemokines contribute to lung inflammation. Chemokine
receptors are expressed on a number of cells, including monocytes, macrophages,
T lymphocytes, eosinophils, and basophils [18]. MCP-1, now termed chemokine
ligand 2 [35, 39], and its receptor CCR2 are potent monocyte and lymphocyte
chemoattractants important in the pathogenesis of several inflammatory diseases,
including atherosclerosis, idiopathic pulmonary fibrosis, and multiple sclerosis [8,
15--18]. Deficiency of CCR2 results in delayed or failed macrophage recruitment,
which in turn causes a reduction in phagocytosis with prolonged influx of and/or
increased numbers of neutrophils and eosinophils and resultant tissue damage [4,
8, 15, 16].
To determine the role of macrophages in MHV-68 infection of the lung,
BALB/c and CCR2)/) mice were infected with MHV-68 and examined histo-
logically. The kinetics of pathologic lesions in the lungs following MHV-68
infection is described. Our data suggest that MHV-68 infection is a useful model
for viral-induced pathology in the lungs and that in the absence of CCR2 there is
increased inflammation.
Macrophages Mediate Viral Lung Pathology 241

Materials and Methods

Animals

Four--six-week-old male BALB/c mice were purchased from Harlan Sprague Dawley, Inc.
(Indianapolis, IN). Six--eight-week-old CCR2)/) mice (BALB/cCmkbr2tm1Kuz) of both genders were a
kind gift from Dr. Gary Hufmagle (University of Michigan, Ann Arbor, MI). All mice were main-
tained in specific-pathogen-free housing and acclimated in the animal facility for 4--7 days before
infection occurred. The mice were housed in accordance with Animal Welfare Act regulations and the
Guide for the Care and Use of Laboratory Animals, under a protocol approved by the University of
Michigan Committee on Use and Care of Animals.

Virus Infection

Generation of MHV-68 stocks was done as previously described [5]. Mice were lightly anesthetized
with isoflurane (AErrane
; Baxter, Deerfield, IL) and inoculated i.n. with 4 · 104 plaque-forming units
(PFUs) of virus, diluted in 20 ll of sterile phosphate buffered saline (PBS; GibcoBRL, Grand Island,
NY). Control mice (mock infected) were inoculated with 20 ll of supernatant from mock virus
preparation at an equivalent dilution in PBS. BALB/c and CCR2)/) mice were euthanized with iso-
flurane at 6 and 12 dpi. Additional BALB/c mice were also euthanized at 16, 20, and 31 dpi.

Ribonuclease Protection Assay

RNA was extracted from tissues as described [6, 24]. MHV-68 gene expression was determined using
the c-7 riboprobe template, as previously described [24]. All riboprobe syntheses were driven by T7
bacteriophage RNA polymerase with [a-32P]UTP (Amersham, Arlington Heights, IL) as the labeling
nucleotide [14]. The ribonuclease protection assay (RPA) was done as described [24]. Bands were
visualized by autoradiography (XAR film, Kodak, Rochester, NY) and were quantified by using the
Storm PhosphorImager and ImageQuant software (Molecular Dynamics, Sunnyvale, CA). Volume
measurements with rectangular objects were used to generate PhosphorImager (PI) counts, which are
presented as a percentage of the internal housekeeping gene signal (i.e., L32) present in each lane.

Histopathology

The left lung was resected, inflated with O.C.T. (Tissue-Tek, Torrance, CA), placed in freezing
embedding medium (O.C.T.), and snap-frozen on dry ice with ethyl alcohol. Frozen tissue was stored
at )80C until sectioning. Serial sections, 4--8 lm thick, were stained with hematoxylin and eosin
(H&E) or prepared for immunohistochemistry.
Upon initial blind evaluation of slides, it was determined that histology findings in uninfected and
mock-infected mice were identical. In both groups, lungs demonstrated minimal focal or no inflammation
and no necrosis or hemorrhage. Consequently, for comparison purposes, lung histology in these two
groups was considered as baseline regarding subsequent evaluation and scoring of infected mice. A score
of 0 reflects no remarkable change. A score of 1 indicates a minimal increase in cells or observation of
necrosis in at least two locations. A score of 2 indicates a mild increase in cell numbers, with at least three
inflammatory cells per high-powered field and/or at least three necrotic cells near a given airway or vessel.
A score of 3 indicates moderate inflammation, with a large contiguous population of cells (30+ cells) and/
or areas of necrosis with at least ten necrotic cells near a given airway or vessel. A score of 4 indicates
marked inflammation, with a high number of inflammatory cells forming clumps, filling the alveoli.
242 J. M. Cadillac et al.

Immunohistochemistry was used to determine which cells responded to MHV-68 infection in

BALB/c and CCR2)/) mice. Cryosections were air-dried for 1--2 hours, fixed in acetone at 4C for 15
minutes, allowed to air dry again, and then stored at )80C [11]. Immunohistochemistry was per-
formed using the Elite ABC Kit (Vector Laboratories, Burlingame, CA) according to the manufac-
turerÕs protocol with several modifications. Buffer of 0.1% saponin in 1 · PBS with 500 mM NaCl was
used throughout the protocol. Sections were incubated with a 1% H2O2 in CH3OH to block endog-
enous peroxidase activity. Sections were then treated with an avidin--biotin blocking complex (Vector
Laboratories), followed by a blocking step using 5% bovine serum albumin (BSA, Vector Laborato-
ries). The primary antibody and controls were then applied and incubated overnight at 4C. Four 15-
minute washes in buffer were performed, followed by application of the diluted biotinylated secondary
antibody solution for 20 minutes. Primary antibodies included antimurine Mac3, Mac1-a, CD16/
CD32, which were all purchased from PharMingen (San Diego, CA); and MOMA-2, which was
purchased from Serotec (Oxford, UK). Mac3, clone M3/84 detects immature monocytes and macro-
phages. Mac1-a, clone CD11b (M1/70) detects granulocytes, macrophages, dendritic cells (DC), and
natural killer (NK) cells. CD16/CD32 detects NK cells, granulocytes, most T cells, monocytes and
macrophages, mast cells, B lymphocytes, and DC cells (at low levels). Rat IgG2a isotype, clone R35--95
(PharMingen) was used for a control antibody. MOMA-2 detects mouse monocytes and macrophages
and rat IgG2b (Serotec) was used for a control antibody. Normal BALB/c mouse serum was a kind gift
from Dr. Jean Nemzek (University of Michigan, Ann Arbor, MI).

Statistics

Group means were compared by two-sample t-test. Statistical significance was set at p < 0.05.

Results

Lung Pathology in BALB/c Mice Infected with MHV-68

To characterize the pathology of the inflammatory response to viral infection,

BALB/c mice were inoculated i.n. with MHV-68 and lungs were processed for
histology or for RNA extraction at 6, 12, 16, 20, and 31 dpi. As shown in Fig-
ure 1, a mild inflammatory response was seen during the first two evaluation time
points (6 and 12 dpi). At both 6 and 12 dpi, there was a perivascular and peri-
bronchial inflammatory pattern characterized by a mixed population of neu-
trophils, lymphocytes, and plasma cells, with an increased alveolar macrophage
population compared with baseline (Fig. 1A, B). By 16 dpi, the perivascular and
peribronchial infiltrates were the most severe (Fig. 1C). There was little hemor-
rhage or necrosis and no alveolar macrophage response, i.e. no continued increase
in numbers of macrophages compared with baseline. At 20 dpi, the inflammatory
response continued to decrease from the preceding time point, with perivascular
and peribronchial infiltrates present to a lesser degree (Fig. 1D). Severity of
inflammation was slightly decreased compared with that at 12 dpi. There was no
increased alveolar macrophage response over baseline at this point, and there was
no hemorrhage or necrosis present. By 31 dpi, the perivascular and peribronchial
inflammation was still evident, but the predominant inflammatory cell types
switched to lymphocytes and a few monocytes (Fig. 1E). This is consistent with
subacute inflammation that classically has a preponderance of lymphocytes and
Macrophages Mediate Viral Lung Pathology 243

variable numbers of monocytes [3]. There was also no alveolar macrophage re-
sponse over baseline and no hemorrhage or necrosis present at 31 dpi.
To quantify the inflammatory response to the virus, H&E-stained sections of
infected lungs at 6 dpi (peak of viral infection) and 12 dpi (following viral
clearance) were evaluated for pathology. Specific criteria were chosen to allow
repeatable scoring based on counting of either inflammatory cells or hemorrhagic
or necrotic foci. Slides were evaluated in six categories: (1) perivascular and
peribronchial inflammation, (2) perivascular necrosis, (3) perivascular hemor-
rhage, (4) increased neutrophils in the alveoli, (5) increased number of alveolar
macrophages, and (6) alveolar necrosis. Each slide was evaluated blindly and was
scored semiquantitatively on a scale from 0 to 4 based on severity of lung
pathology (see the Methods section for scoring explantation). With the exception
of mean neutrophil numbers, mean scores of inflammation, necrosis, and hem-
orrhage increased between 6 and 12 dpi (Fig. 2A).
The lungs of infected BALB/c mice had intense positive staining for mono-
cytes and macrophages with the macrophage markers MOMA-2 (Fig. 3A) and
Mac3 (data not shown) at 6 dpi and light positive staining by 12 dpi. These
immunohistochemistry results confirm the presence of monocytes and macro-
phages in MHV-68 infection during both acute and recovered infection as noted
by histologic analysis.
To verify viral infection of the mice, RNA was analyzed by RPA to measure
expression of viral transcripts. We observed high-level viral gene expression at 6
dpi (Fig. 4) consistent with our previous observations that 6 dpi corresponds to
the acute phase of viral infection in the lung [38]. No viral gene expression was
observed at the later time points (Fig. 4 and data not shown) verifying that virus
was cleared from the lungs by 12 dpi.

Role of CCR2 in MHV-68-Induced Inflammatory Response

CCR2 is the receptor for the monocyte chemoattractant protein-1 (MCP-1) [39].
Infection of mice deleted of CCR2 (CCR2)/) mice) results in reduced macrophage
recruitment in the lungs following viral infection [16]. To determine if the absence
of CCR2 would impact the pathogenesis of MHV-68 infection, CCR2)/) mice
were infected i.n. with MHV-68 and lungs were processed for histology or for
RNA extraction. We observed a robust pattern of viral gene expression in the
CCR2)/) infected mice at 6 dpi as determined by RPA analysis (Fig. 4). Viral
gene expression was no longer detectable by 12 dpi in the CCR2)/) mice, indi-
cating that the virus was cleared by this time point. No significant differences in
the pattern of viral gene expression were observed following infection of BALB/c
or CCR2)/) mice (Fig. 4B).
In the CCR2)/) infected mice, inflammation was characterized most com-
monly by a mixed infiltrate of neutrophils, lymphocytes, and plasma cells. Peri-
vascular inflammation and peribronchial inflammation were minimal in CCR2)/)
mice at 6 dpi (Fig. 5A, B), and the mean score for alveolar macrophage accu-
mulation was less than 1 (Fig. 2A). By 12 dpi, scores for all six evaluation cate-
244 J. M. Cadillac et al.

Fig. 1. Characterization of lung pathology following intranasal infection with MHV-68 in the lungs of
BALB/c mice. All sections are prepared from frozen tissue and stained with H&E. (A) Acute infection
(6 dpi) characterized by a mild mixed-cell inflammatory infiltrate which caused an increased lung
cellularity; scale = 200 lm. Inset shows accumulation of neutrophils (arrowheads) and lymphocytes.
(B) By 12 dpi, a moderate mixed-cell inflammatory response is seen with an increased alveolar mac-
rophage population; scale = 200 lm. Inset shows neutrophils (arrowheads) and alveolar macro-
phages (arrows). (C) At 16 dpi, the mixed-cell infiltrate is most severe, characterized by multifocal
dense accumulations of inflammatory cells; scale = 200 lm. Inset shows cellular infiltrates in the
peribronchial regions. (D) By 20 dpi, the inflammatory response has greatly decreased compared with
the earlier time points; scale = 200 lm. (E) At 31 dpi, a diffuse lymphocytic infiltrate is seen; sca-
le = 200 lm. Inset shows dense accumulation of lymphocytes (arrows). Original magnifications: · 200
(A--E); insets · 400 (A, B, E), · 800 (C).
Macrophages Mediate Viral Lung Pathology 245

Fig. 2. Mean histology

scores from lungs of
BALB/c and CCR2)/)
mice infected i.n. with
MHV-68. (A) Overall
average histology scores
graded from 0 (no change)
to 4 (marked
inflammation). BALB/c
and CCR2)/) infected mice
were compared at 6 dpi
and 12 dpi in six
categories: (B, C) Lanes 1--
6 represent pathology
categories as indicated: 1,
perivascular and
peribronchial
inflammation; 2,
perivascular necrosis; 3
perivascular hemorrhage; 4
increased neutrophils in
the alveoli; 5, increased
number of alveolar
macrophages; and 6,
alveolar necrosis. Each
data point represents the
average of 3--4 mice per
group for both 6 and 12
dpi. *p < 0.05.
246 J. M. Cadillac et al.

Fig. 3. Evaluation of the inflammatory response to MHV-68 infection in the lungs of BALB/c and
CCR2)/) mice using immunohistochemistry on frozen sections. (A) MOMA-2 positive staining of
alveolar macrophages (arrows) at 6 dpi in a BALB/c mouse, showing numerous macrophages. (B) Few
macrophages are seen at 6 dpi in a CCR2)/) mouse, as expected with a deficiency in CCR2. Original
magnifications: · 400.

gories (Fig. 2C) were increased from the prior time point (Fig. 2B). Overall his-
tology scores for BALB/c and CCR2)/) mice show a trend of increasing
inflammation from 6 dpi to 12 dpi. At 12 dpi (Fig. 2C), there is a significant
difference between the BALB/c mice and the CCR2)/) mice when comparing
perivascular necrosis and diffuse alveolar neutrophil infiltrates (p < 0.05).
CCR2)/) mice at both time points had lower mean alveolar macrophage scores
(i.e., less macrophage accumulation) when compared with BALB/c mice.
By 12 dpi (Fig. 5C), the CCR2)/) mice showed increased perivascular
necrosis and increased numbers of neutrophils in the alveoli that were significantly
greater when compared with those of the BALB/c mice (p < 0.05). Perivascular
and peribronchial regions of hemorrhage were observed in both the BALB/c and
the CCR2)/) mice, associated with regions of severe perivascular and peribron-
chial necrosis and inflammation. Necrosis of the alveolar epithelial cell lining was
a rare finding, noted only in CCR2)/) animals (one mouse at 6 dpi and one mouse
at 12 dpi).
At 6 and 12 dpi, lungs of CCR2)/) mice showed light positive staining for
monocytes and macrophages with MOMA-2 (Fig. 3B) and Mac3 (data not
shown). These results support the histologic observations that there were a de-
creased number of alveolar macrophages present in the CCR2)/) mice compared
with the BALB/c mice.

Summary of Histological Findings

Inflammation, necrosis, and increases in alveolar macrophages were more severe

at 12 dpi than at 6 dpi for both BALB/c and CCR2)/) mice. CCR2)/) mice
showed greater inflammation at 12 dpi than did BALB/c mice. This inflammation
was accompanied by statistically significant higher incidence and severity of
Macrophages Mediate Viral Lung Pathology 247
248 J. M. Cadillac et al.

b
Fig. 4. (A) Viral gene expression in lungs of CCR2)/) mice infected with MHV-68. RNA was har-
vested at 6 and 12 dpi, and 5 lg of RNA was analyzed for expression of MHV-68 transcripts by RPA
with the c-7 probe set. Shown is a representative phosphorimage. Each lane represents RNA extracted
from a single mouse. (B) Quantitation of MHV-68 gene expression following infection of BALB/c or
CCR2)/) mice. PI counts were obtained for protected probe fragments and the data are presented as a
percentage of the internal housekeeping signal L32. Means ± SEM are shown for each time point for
balb/c and CCR2-/- mice.

perivascular necrosis and diffuse alveolar neutrophil infiltrates in CCR2)/) mice,

possibly secondary to decreased alveolar macrophage recruitment in these
animals.

Discussion

MHV-68 provides a useful model system to study the pathogenesis of gam-

maherpesviruses and host responses to this viral infection. In this study, we have
characterized the pathogenesis of MHV-68 in BALB/c and CCR2)/) mice,
focusing on the histopathologic features of infection, identification of cells in-
volved, and the role of CCR2 involvement in macrophage recruitment during
viral infection.
We found that lung pathology in BALB/c mice infected with MHV-68 was
characterized by a mixed inflammatory infiltrate, necrosis, and increased alveolar
macrophages by 12 dpi consistent with previous studies [22, 30]. In many lung
sections from the CCR2)/) mice, approximately 10--15% of nuclei in perivascular
regions were apoptotic. This far exceeds the expected percent of inflammatory
cells which may undergo apoptosis in a typical inflammatory reaction and sup-
ports the fact that resident epithelial and stromal cells in perivascular regions are
undergoing necrosis. Inflammation was still present at 30 dpi, consisting primarily
of lymphocytes and a few monocytes. This contrasts with previous studies that
reported only persistent areas of lymphoid cells in the lungs [22, 30]. The presence
of both lymphocytes and monocytes is more typical of subacute to chronic
inflammation [3, 23], suggesting that MHV-68 infection, while resolved by 12 dpi,
initiates a chronic inflammatory stimulus. Recent studies show that low-level
infectious MHV-68 can be isolated from the lungs greater than 30 dpi [12] and it is
possible that this low-level persistence of the virus could maintain this inflam-
matory stimulus.
At 6 dpi, CCR2)/) infected mice demonstrated fewer alveolar macrophages
with minimal perivascular and peribronchial inflammation compared with
BALB/c infected mice. By 12 dpi, the CCR2)/) infected mice showed a statisti-
cally significantly higher severity of perivascular necrosis and diffuse alveolar
neutrophil infiltrates and a high incidence of peribronchial necrosis. Immuno-
histochemistry results confirm the presence of fewer monocytes and macrophages
in CCR2)/) mice than in BALB/c mice. This is consistent with the prolonged
neutrophil response as a result of decreased phagocytosis due to delayed mac-
rophage migration that has been reported in CCR2)/) mice [8, 21]. Neutrophils
Macrophages Mediate Viral Lung Pathology 249

Fig. 5. Inflammatory response to MHV-68 in lungs of CCR2)/) mice. Frozen sections were stained,
with H&E. (A) Perivascular inflammation and hemorrhage in the lungs of a CCR2)/) mouse at 6 dpi,
scale = 200 lm. Inset shows numerous lymphocytes (arrows) and alveolar macrophages (arrow-
heads). An area of hemorrhage is present between the two labeled macrophages. Several cells in the
vessel wall are necrotic. (B) Higher magnification of a CCR2)/) mouse at 6 dpi showing several
picnotic cells which characterize perivascular and alveolar wall necrosis (arrows); scale = 50 lm.
There are multiple neutrophils also identified. (C) Perivascular infiltrates and necrosis in a CCR2)/)
mouse at 12 dpi; scale = 200 lm. Lung interstitium and alveolar spaces are highly cellular. Area
between arrows has increased numbers of neutrophils and alveolar macrophages with numerous
lymphocytes also present. Original magnifications: ·400 (A, C), · 700 (B).

typically are predominant in acute inflammation [3], while neutrophils, lympho-

cytes, and plasma cells can all be seen in subacute inflammation [3].
Altogether, these findings suggest that CCR2 is important in monocyte traf-
ficking during inflammation and infection with MHV-68. Several investigators have
shown that CCR2)/) mice challenged immunologically with various pathogens and
nonspecific inflammatory stimuli demonstrate defects in macrophage function [8,
15--17, 19]. These results demonstrate that macrophages are also important mod-
ulators of inflammation in this murine model of gammaherpesvirus-induced lung
pathology as the lung pathology is more severe in the absence of macrophages.
Understanding which cell populations are associated with the host response to viral
infection in the lungs and determining the long-term effect of viral infection on
chronic inflammatory responses can hopefully lead to interventions for the pre-
vention and treatment of viral-induced lung disease.
250 J. M. Cadillac et al.

Acknowledgments. This work was supported in part by NIH/NCRR T32 RR07008 (J.C.), NICHD
grant HD28820 (J.W.), and NIA AG908692 (R.R.). The authors would like to thank the Martin
Philbert laboratory for use of their cryostat, Thomas Komorowski for his technical assistance, and Dr.
Ken Guire, University of Michigan, and Dr. Weidong Zhang, The Jackson Laboratory, for advice on
statistical analysis. The authors also thank Elizabeth Horn and Mark Deming from Pathology Pho-
tography for their technical assistance. The authors are especially grateful to the personnel at Diane
Robins Laboratory for their support.

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Accepted for publication: January 3 2005