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From the Intestinal Disease Research Programme, Department of Pathology and Molecular Medicine, McMaster University, Hamilton, Ontario, Canada
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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(TNF-) levels, and epithelial ion transport (baseline and
stimulated by electrical nerve stimulation, carbachol,
and forskolin) were examined. DSS-treated mice displayed a variable
diarrhea, significant histopathology in the mid-distal
colon, elevated MPO activity, and reduced (>50%)
responses to all three pro-secretory stimuli. Treatment with
rolipram, and to a lesser extent pentoxifylline,
significantly reduced the severity of the colonic histopathology and
MPO levels. Neither PDE inhibitor had any affect on the diminished ion
transport events caused by DSS-induced colitis. However,
although stimulated ion transport events were still reduced 3 days
after DSS treatment, colonic segments from DSS +
rolipram-treated mice displayed enhanced recovery in their secretory
responsiveness, particularly to carbachol. These findings
indicate that specific PDE4 inhibition can significantly reduce the
tissue damage that accompanies colitis and enhance recovery of normal
colonic function.
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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on
stimulation with bacterial endotoxin.2
cAMP is hydrolyzed
to the inactive 5'AMP form by members of a superfamily of enzymes, the
phosphodiesterases (PDE), of which type 4 (PDE4) is the predominant
form in immune cells.1,3,4
Thus, inhibition of PDE
activity that would result in maintained elevation of cAMP levels in
immune cells presents itself as a potential anti-inflammatory or
immunosuppressive strategy.5,6
In fact, numerous in vitro
studies have documented that nonselective PDE, or specific PDE4
inhibitors, down-regulate the activity of most types of immune cells,
including T-cells, macrophages, neutrophils, and mast
cells.1,3
Similarly, PDE inhibitors have been found to be
beneficial in animal models of airway or joint
inflammation.7,8
In the latter scenario, it was suggested
that the amelioration of collagen-induced arthritis in mice by the PDE4
inhibitor, rolipram (ROL), was because of suppression of TNF-
production. In this context, it is noteworthy that neutralizing TNF-
antibodies are an effective short-term treatment for a cohort of
steroid-resistant patients with Crohn’s disease.9
However, the ability of inhibitors of PDE activity to alleviate either
the functional abnormalities or histopathology associated with enteric
inflammation has not been extensively examined.
Oral administration of dextran sulfate sodium (DSS) to rodents results
in overt inflammation in the mid-distal colon that is somewhat
reminiscent of human inflammatory bowel disease. Exposure to DSS leads
to a time- and dose-dependent drop in body weight, a variable
watery/bloody diarrhea, and can result in rectal prolapse and
fatality.10,11
A number of approaches have been used to
reduce the severity of DSS-induced histopathology in the colon, such as
treatment with ICAM-1 anti-sense oligonucleotides,12
recombinant IL-10,13
inhibition of 5-lipoxygenase or
neutrophil activity,14,15
and neutralization of
TNF-.16
However, considerably fewer studies have
examined changes in colonic physiology as a consequence of exposure to
DSS.
The present study was designed to compare the capacities of the nonselective PDE inhibitor, pentoxifylline (PTX), and the PDE4 inhibitor, ROL, to affect gut inflammation as judged by both structure and epithelial ion transport in the DSS model of murine colitis. The data show that concomitant twice-daily treatment with ROL, and to a lesser extent PTX, significantly reduced the severity of the colonic histopathology induced by a 5-day course of DSS drinking water. However, the disrupted epithelial ion transport apparent at the end of the DSS-treatment period was not affected by either inhibitor of PDE activity.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Male BALB/c mice (6 to 8 weeks old) were purchased from Harlan Animal Suppliers (Indianapolis, IN) and were housed under conventional conditions with free access to animal chow and water. For the induction of colitis, normal drinking water was replaced with a 4% (w/v) solution of DSS (molecular weight, 40 kd; ICN Biomedicals Inc., Aurora, OH) for 5 days.17 PTX or ROL (both from Sigma Chemical Co., St. Louis, MO) were administered twice daily, intraperitoneally (i.p.) at a dose of 5 mg/kg body weight in 200 µl of phosphate-buffered saline (PBS) beginning on the day of DSS exposure.7,8,18,19 Pilot studies examined the effect of a single daily injection of ROL at the same dose. Time-matched controls consisted of naïve mice, mice administered DSS, ROL, or PTX only, and those injected with ethanol (10 µl in 190 µl of PBS) only, the vehicle for ROL. All experiments were approved by the Animal Care Committee at McMaster University.
Macroscopic Assessment
Animals were weighed and their behavior observed daily. On day 5,
water intake was recorded (expressed as ml/day/mouse), mice were
sacrificed by cervical dislocation, and the entire colon (from
ileocecal junction to the anus) was excised. Colon length was measured
and observations regarding ulceration, vascularization, and stool
consistency were recorded (Table 1)
.
Previous studies have shown colonic shortening during
colitis,10
and so the colon was divided based on total
length: the mid-distal portion (ie, 30 to 60% region) was used for
electrophysiology studies; the adjacent distal 10% was fixed for
histological examination; and the remainder of the tissue was
snap-frozen in liquid N2 before processing for
myeloperoxidase (MPO) activity or TNF- levels.
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Tissue was fixed in 10% neutral buffered formalin, dehydrated, and wax-embedded. Five-µm sections were collected on coded slides, stained with hematoxylin and eosin, and scored in a blinded fashion by two investigators. Histological damage was scored using the criteria of Appleyard and Wallace20 which considers loss of mucosal architecture, cellular infiltration, muscle thickening, crypt abscess formation, and goblet cell depletion (maximum score = 11).
MPO Activity
MPO activity was determined following a published protocol.21 Briefly, tissue samples were weighed and suspended in 50 mmol/L potassium phosphate buffer (pH 6.0) containing 5 mg/ml hexadecyltrimethylammonium bromide (Sigma Chemical Co.) at a ratio of 50 mg tissue to 1 ml of buffer. Tissues were homogenized by a polytron tissue homogenizer for 15 seconds, and 1 ml was decanted into sterile Eppendorf tubes and centrifuged at 12,000 rpm for 15 minutes. Using a microtiter plate scanner, 200 µl of the reaction mixture (containing 16.7 mg of o-dianisidine (Sigma Chemical Co.), 90 ml of distilled H2O, 10 ml of potassium-phosphate buffer, and 50 µl of 1% H2O2) was added to each well containing 7 µl of sample in a standard 96-well plate and three absorbance readings at 30-second intervals at 450 nm were recorded. MPO activity was measured in units/mg tissue, where one unit of MPO was defined as the amount needed to degrade 1 µmol of H2O2 per minute at room temperature.
Functional Studies
Epithelial ion transport was examined in colonic segments (approximately mid-distal colon) mounted in Ussing chambers.22 Tissues (surface area = 0.6 cm2) were bathed in 10 ml of warm (37°C), oxygenated Krebs buffer (pH 7.35 ± 0.02). The spontaneous potential difference was maintained at 0 mV by an automated voltage clamp (WPI, Mississauga, Ontario, Canada), and the short-circuit current (Isc in µA/cm2) was continuously measured as an indicator of net active ion transport. At 15-minute intervals, tissue conductance (G; indicates barrier to passive ion flow) was calculated from Isc and potential difference values.
Stimulated ion transport events were sequentially evoked by: 1)
electrical transmural stimulation (10 Hz, 10 mA, 0.5 ms for a total
time of 5 seconds); 2) addition of the cholinergic agonist, carbachol
(10-4 mol/L; Sigma Chemical Co.) to the
serosal buffer; and 3) addition of the adenylate cyclase-activating
agent, forskolin (10-5 mol/L; Sigma
Chemical Co.) to the serosal buffer.23
In all
instances, the effect of the treatment was recorded as the
maximum change in Isc (ie, 
Isc) to occur within 5 minutes.
Cytokine Production
At the end of the DSS-treatment period, blood was collected for
determination of serum TNF- levels by enzyme-linked immunosorbent
assay using paired antibodies from PharMingen Inc. (detection limit 32
pg/ml; Mississauga, Canada). Portions of terminal colon were
homogenized in PBS containing 2 mmol/L of phenyl-methyl sulfonyl
fluoride (Sigma Chemical Co.) and tissue levels of TNF- were
measured. All cytokine determinations were performed in duplicate
serial dilutions.
In Vitro Analysis of DSS Epithelial Toxicity
The murine IEC-4.1 epithelial cell line was seeded in 12-well sterile plates (106 cells/well) and grown for 24 hours under standard culture conditions24 and then exposed to 1% or 2% DSS ± ROL (10-6 mol/L). Twenty hours later the enterocytes were retrieved by addition of trypsin/ethylenediaminetetraacetic acid and cell viability determined using the trypan blue (0.04% w/v) dye exclusion technique. In another set of experiments, enterocytes were seeded into 96-well plates (3.5 x 104 cells/well) and 24 hours later were exposed to 1% or 2% DSS ± ROL for 20 hours. Subsequently each well was pulsed with 50 µg of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide; Sigma Chemical Co.) for 5.5 hours followed by the addition of 50 µl of 10% (v/v) Triton-X 100/0.5 mol/L HCl and a 24-hour incubation in the dark. Optical density was measured at 540 nm.25
Recovery Experiments
In a final series of experiments, mice were exposed to 4% DSS for 5 days with or without daily ROL (5 mg/kg, twice daily). Subsequently, the animals received normal drinking water for 3 days (ROL was not administered during this period), and were then autopsied and the colonic form and function were assessed.
Analysis and Data Presentation
All data are expressed as means ± SEM (SEM), where n refers to the number of mice in each experiment. Data were compared using one-way analysis of variance (WINKS software by Texsoft, Cedarhill, TX) and P < 0.05 was accepted as the level of statistically significant difference compared to time-matched controls.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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. However, the beneficial effect of ROL
(twice daily) described below is unlikely to be because of the reduced
DSS intake because mice receiving ROL once a day developed a colitis
virtually indistinguishable from that in the DSS-positive control
group, despite their reduced DSS water intake (ie, histopathology
damage scores were not significantly different when DSS and DSS + ROL,
once a day, were compared).
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After 5 days of ad libitum exposure to 4% DSS many of
the mice displayed clinical and macroscopic signs of inflammation
and/or gut dysfunction. The distal two-thirds of the colon of
DSS-treated mice was consistently devoid of contents, whereas the colon
of DSS + ROL-treated mice typically contained loosely formed stool.
Table 1
shows that all animals receiving DSS, independent of PDE
treatment displayed some signs of diarrhea or perturbed water balance;
however, luminal fluid content was not quantified. Typical of other
colitidies, the DSS-treated animals had a significantly shortened
colon, which was partially prevented by ROL and PTX (Table 1)
. In
addition, DSS treatment resulted in a significant drop in body weight
that was not abrogated by PTX and was exaggerated by concomitant ROL
therapy (Table 1)
. This additional drop in body weight may be a
consequence of the reduced water intake in this group of mice.
Histology
Figure 2, a and b
, shows normal
colonic structure and that ROL treatment (twice daily) did not alter
colonic architecture, respectively. Colitis evoked by DSS was
characterized by severe disruption of tissue architecture, edema, a
massive mixed immune cell infiltrate (mononuclear cells, neutrophils,
and eosinophils), ulceration and significant areas of complete
epithelial denudation, and muscle thickening (Figure 2c)
. Mice treated
with ROL once daily along with DSS experienced the same fate as those
in the DSS only group (data not shown). However, animals treated with
ROL twice daily showed a significant improvement in colonic histology,
ranging from an appearance virtually indistinguishable from control
tissue, to colons showing a mild immune cell infiltrate (Figure 2d)
,
and those showing small foci of architectural destruction. Comparison
of the tissue damage scores showed that colons from DSS-treated mice
had scores in the range of 5 to 11 (11 is maximum), whereas 11 of 12
animals in the DSS + ROL (twice daily) group had scores of <5; colon
from the remaining animal in this group had a damage score of 5 (Figure 3)
. This reduction in colonic damage is
complemented by the reduction in the number of mice displaying evidence
of colonic/rectal bleeding (Table 1)
. Mice treated with DSS + PTX
displayed an improvement in colonic histopathology compared to DSS-only
mice, but there were still clear signs of edema, inflammatory
infiltrate, and muscle thickening (Figure 2e)
. The damage score of DSS
+ PTX-treated mice was intermediate between DSS-only and DSS +
ROL-treated mice (Figure 3)
.
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Measurement of MPO activity revealed that DSS colitis was accompanied by an increase in MPO and that this was significantly reduced by concomitant ROL or PTX (twice daily): control = 0.10 ± 0.19; DSS = 1.53 ± 0.3 (P < 0.05 compared to control, n = 9 to 20); DSS + ROL = 0.13 ± 0.21; and DSS + PTX = 0.38 ± 0.23 U/mg wet weight of tissue.
Epithelial Function
Baseline Isc, potential difference, and tissue conductance values
are shown in Table 2
. These gut
parameters were not significantly different when tissues from control
and DSS colitis mice were compared. In contrast, responses to all three
pro-secretory stimuli were significantly reduced in tissues from mice
treated with DSS, and this diminished responsiveness was not affected
by co-treatment with ROL or PTX (twice daily) (Figure 4)
.
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Enzyme-linked immunosorbent assay of serum samples
(n = 6) and tissue homogenates
(n = 3) revealed no detectable TNF- in any of
the treatment groups. (The assay protocol used herein has been used to
successfully detect serum levels of TNF- in other studies in our
laboratory.)
Epithelial Viability
As shown in Figure 5
, exposure of a
murine epithelial cell line to 1% or 2% DSS for 20 hours caused a
two- to threefold increase in cell death that was not altered by
concomitant treatment with ROL (10-6
mol/L). Similarly, use of the MTT assay as an indicator of epithelial
viability (specifically mitochondrial function), revealed a significant
reduction in MTT metabolism in epithelial preparations exposed to DSS
that was not abrogated by concomitant ROL treatment: control =
0.71 ± 0.04; 1% DSS = 0.58 ± 0.03*, 1% DSS +
ROL = 0.58 ± 0.02*, 2% DSS = 0.52 ± 0.0.02*, 2%
DSS + ROL = 0.57 ± 0.04* optical density (OD) units
(n = 8 replicates from a representative
experiment, 3 experiments were performed; *P < 0.05
compared to control; cells treated with distilled water as a positive
control = 0.05 ± 0.01* OD units).
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The ability of ROL to reduce the histopathology in DSS colitis
suggested the possibility that ROL-treated mice might recover more
quickly from a 5-day course of DSS. In accordance with previous
histological data, ROL + DSS-treated mice examined 3 days after the end
of the treatment showed a significant preservation of colonic structure
(Figure 2f)
: damage scores, DSS only = 6.5 ± 0.9 and ROL +
DSS = 1.9 ± 0.3 (n = 3 and
n = 5, respectively). Stimulated epithelial ion
transport events in colonic segments from mice 3 days after withdrawal
of DSS were significantly reduced compared to control tissues (Table 3)
and this reduction was similar to, or
of a greater magnitude than, that observed from colonic tissue excised
at the end of the 5-day DSS treatment (compare Figure 4
and Table 3
).
However, colon from mice treated with ROL + DSS showed a partial or
statistically significant improvement in their responsiveness to
pro-secretory stimuli (Table 3)
. In addition, carbachol challenge of
colon excised from mice 3 days after the DSS treatment resulted in a
drop in Isc (shown as a negative value in Table 3
), rather than the
expected transient increase in Isc that occurs in tissue from normal
mice. In contrast, tissues from mice treated with DSS + ROL all
displayed an increase in Isc in response to carbachol, although the
magnitude of the response was very variable (6.7 to 65.5
µA/cm2).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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synthesis as a key component of
their anti-inflammatory activity.2,5-7
These data suggest
that inhibition of PDE activity could be a rational therapy for human
idiopathic inflammatory bowel disease, disorders that are often
accompanied by elevated TNF- levels.9
Indeed,
preliminary data from animal studies18,26
and a trial
involving seven patients27
support this postulate.
However, evaluations of the use of PDE inhibitors to treat enteric
inflammation have focused on histopathology and primarily neglected
assessment of gut function. This study was designed to examine the
ability of a PDE4-selective (ie, ROL) and a nonselective PDE inhibitor
(ie, PTX) to modulate the structural and functional abnormalities in
murine colitis. Thus, using the established DSS model of murine
colitis,10,11
we show that: 1) ROL is more effective than
PTX in preventing the DSS-induced colonic histopathology; 2) neither
inhibitor affected the ion transport irregularities observed at the end
of a 5-day DSS treatment; and 3) ROL-treated mice showed a more rapid
recovery in their responsiveness to pro-secretory agents compared to
DSS-only treated mice. Steroids are a mainstay in the treatment of inflammatory bowel disease and although they are very effective, their long-term use results in considerable side effects. Hence, there is a need for the development of other therapeutics to combat enteric inflammation, and one such strategy is to enhance immunosuppression by maintaining elevated cAMP levels via inhibition of PDE activity. Twice-daily treatment of mice with low-dose ROL or PTX resulted in significantly less colonic histopathology in animals with free access to 4% (w/v) DSS drinking water. Thus, general inhibition of PDE activity by PTX, or specific targeting of PDE4 by ROL resulted in greater preservation of colonic structure, and a concomitant decrease in tissue MPO levels compared to mice receiving DSS and saline or DSS and drug vehicle. PTX was consistently less effective than ROL in abrogating colonic pathology and because it was used at the same concentration as ROL, a value 20 times less than that used in other PTX studies,26,28 this suggests that the benefit of either drug is because of inhibition of PDE4, the predominant PDE in immune cells. These data support the use of PDE4 inhibitors as an anti-inflammatory option and confirm recent data showing that inhibition of PDE activity reduces the histopathology associated with colitis.18,26-28
Analysis of colonic epithelial ion transport in the DSS colitis model
has hitherto not been reported. Many of the mice exposed to DSS
displayed a variable diarrhea and the colons of all treated animals
were inflamed, often with significant epithelial loss. It is noteworthy
that the ROL-treated (twice daily) animals, although showing
histological improvement still had macroscopic signs of diarrhea/water
imbalance (Table 1)
and this treatment did not ameliorate the ion
transport (create the driving force for directed water movement)
abnormalities caused by exposure to DSS (see below). Baseline Isc
across colonic tissue from all DSS-treated mice (regardless of
concomitant PDE therapy), was not consistently elevated and ion
conductance was within the range for normal tissue, suggesting
unaltered permeability. Neither observation fits with the obvious
tissue pathology. However, Isc is a composite of all of the active ion
transport across the tissue and so additional studies examining
specific ion movements in the colitic, and adjacent tissue are required
before precise statements regarding the driving forces for water
movement can be made. Also, it is inconceivable that epithelial loss
would not result in increased permeability and indeed DSS-induced
colitis has been shown to increase permeability.29,30
The
absence of a significant increase in conductance observed here is
likely because of the use of whole-thickness tissues in the Ussing
chamber, such that any increase in epithelial permeability was offset
by muscle hypertrophy (compare Figure 2, a and c
).
Increases in Isc evoked by all three pro-secretory stimuli were significantly reduced in colonic segments excised from DSS-treated mice. Similar diminished Isc responsiveness has been observed in other animal models of colitis and in tissue resections from patients with inflammatory bowel disease.31-34 However, neither ROL nor PTX treatment led to any amelioration of the reduced Isc responses, clearly indicating uncoupling of structure and function in this model of colitis. Juxtaposition of these data with the histological portion of the study suggests that either regulation of Isc is more readily altered than derangement of colon structure, or that ion transport and colon structure are regulated by different mechanisms. Similarly, we and others have shown that colonic ion transport can be perturbed in the absence of any evidence of histological abnormalities as defined by light microscopy.22,32 For instance, colon excised from rats treated with the pro-colitic haptenizing agent TNBS (2,4,6-trinitrobenzenesulfonic acid) seems structurally normal 12 weeks after treatment, and although baseline ion transport and tissue conductance were within the normal range, responsiveness to nerve stimulation and carbachol remained depressed compared to tissues excised from controls.32 Thus, in assessing the value of any putative anti-inflammatory agent it would seem imperative that structural analysis be complemented by functional studies.
Given the ability of ROL to almost completely prevent DSS-induced histopathology, we postulated that ROL therapy might hasten the recovery process after DSS exposure. In support of this, we observed that whereas colonic tissue from DSS-only treated mice displayed reduced Isc responses to electrical nerve stimulation, carbachol and forskolin 3 days after replacing the DSS water with regular water, the colon from the ROL + DSS-treated animals showed a significant recovery in their secretory responsiveness. These data add further credence to the hypothesis that PDE inhibition may be a valuable adjunct therapy for colonic inflammation.
The mechanism responsible for DSS-induced colitis is unknown and a
variety of anti-inflammatory strategies have been used to treat the
colitis, at least in terms of the
histopathology.12-16,29,35,36
Furthermore, the induction
of colitis in severe combined immunodeficient,
CD4+-T cell depleted, and athymic mice by DSS
reveals that thymus-dependent T cells are not a prerequisite for the
colitis37,38
This has led to the suggestions that
DSS-induced colitis is either a macrophage-driven
event39,40
(which fits with a pivotal role for TNF-),
or because of direct epithelial cytotoxicity/inhibition of
proliferation.37,41,42
Thus, we sought to examine the
affect of ROL therapy on TNF- levels and epithelial viability.
At the end of the 5-day DSS exposure, TNF- levels were neither
detected in the serum nor colonic tissue homogenates from control, DSS,
or DSS + ROL-treated mice. Thus, we can provide no evidence in support
of inhibition of TNF- production as a component of the
anti-inflammatory activity of ROL in this model system. However,
because TNF- is rapidly mobilized it is possible that ROL did reduce
the levels of TNF- earlier in the treatment regimen. Moreover,
although numerous studies have shown that phosphodiesterase (PDE)
inhibition reduces macrophage-stimulated TNF-
production,2,7
it has recently been shown that the
reduction in nonsteroidal anti-inflammatory drug-induced
enteropathy in the rat by ROL was independent of it’s ability to
reduce plasma TNF- levels.43
Also, the clinical
improvement in patients with Crohn’s disease treated with PTX did not
correlate with reduced tissue TNF- levels.27
In
addition, PDE inhibition can result in reduced IL-1ß, IL-6, IL-8, and
IL-12 levels and clearly any, or all of these cytokines may be involved
in mediating enteric inflammation.44-46
Using the vital stain, trypan blue, and the MTT assay to assess epithelial viability, we found that DSS was directly toxic to the murine IEC.4.1 cell line in vitro. These findings are in accordance with other publications.37 However, simultaneous ROL treatment did not abrogate the DSS cytotoxic effects and we speculate that the beneficial effect of ROL in vivo is not because of inhibition of epithelial cell death by directly affecting the epithelial cell.
In summary, we have shown that use of the PDE inhibitors ROL and PTX significantly reduced the histopathology in a murine model of colitis. Neither agent affected the reduced secretory responsiveness that was apparent in colonic tissues examined at the end of the 5-day DSS-treatment period, although ROL therapy did enhance the recovery phase after withdrawal of the colitic stimulus. These findings add to the list of models of colitis that are characterized by perturbations in ion transport and complement a smaller number of investigations illustrating that normal histological appearance is not necessarily reflective of functional normality. We suggest that PDE inhibitors, particularly those targeted at the PDE4 isoform,47 warrant further assessment as tools to combat enteric inflammation either alone or as part of a combination therapy regimen.28
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Acknowledgements |
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Footnotes |
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Supported by grants from the Crohn’s and Colitis Foundation of Canada and the Medical Research Council of Canada (# MT-13421) (to D. M. M.).
This work was presented in part, in abstract form, at the Pan-American Congress of Gastroenterology in August 1999 at Vancouver.
Accepted for publication February 16, 2000.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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expression, inhibition of Th1 activity, and amelioration of collagen-induced arthritis by rolipram. J Immunol 1997, 159:6253-6259[Abstract]
for Crohn’s disease. N Engl J Med 1997, 337:1029-1035
and tumour necrosis factor-. Inhibition of increased permeability, but not diminished secretory responses by transforming growth factor ß2. J Immunol 1997, 159:2382-2390 monoclonal antibody in dextran sulfate-induced mouse colitis. Aliment Pharmacol Ther 1999, 13:251-260[Medline]
release. Am J Physiol 1999, 277:G847-G854 by human peripheral blood mononuclear cells. Immunology 1994, 83:262-267[Medline]
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, statistically different
(P <
0.05) groups compared to each other and control
(CON). See Figure 1
