Rotavirus Infection Increases Intestinal Motility
but Not Permeability at the Onset of Diarrhea
Claudia Istrate, Marie Hagbom, Elena Vikström, Karl-Eric Magnusson and Lennart Svensson
Linköping University Post Print
N.B.: When citing this work, cite the original article.
Original Publication:
Claudia Istrate, Marie Hagbom, Elena Vikström, Karl-Eric Magnusson and Lennart Svensson,
Rotavirus Infection Increases Intestinal Motility but Not Permeability at the Onset of
Diarrhea, 2014, Journal of Virology, (88), 6, 3161-3169.
http://dx.doi.org/10.1128/JVI.02927-13
Copyright: American Society for Microbiology
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Postprint available at: Linköping University Electronic Press
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Rotavirus Infection Increases Intestinal
Motility but Not Permeability at the Onset of
Diarrhea
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Claudia Istrate, Marie Hagbom, Elena Vikström, Karl-Eric
Magnusson and Lennart Svensson
J. Virol. 2014, 88(6):3161. DOI: 10.1128/JVI.02927-13.
Published Ahead of Print 26 December 2013.
Rotavirus Infection Increases Intestinal Motility but Not Permeability
at the Onset of Diarrhea
Claudia Istrate,a,b Marie Hagbom,b Elena Vikström,c Karl-Eric Magnusson,c Lennart Svenssonb
ABSTRACT
The disease mechanisms associated with onset and secondary effects of rotavirus (RV) diarrhea remain to be determined and
may not be identical. In this study, we investigated whether onset of RV diarrhea is associated with increased intestinal permeability and/or motility. To study the transit time, fluorescent fluorescein isothiocyanate (FITC)-dextran was given to RV-infected
adult and infant mice. Intestinal motility was also studied with an opioid receptor agonist (loperamide) and a muscarinic receptor antagonist (atropine). To investigate whether RV increases permeability at the onset of diarrhea, fluorescent 4- and 10-kDa
dextran doses were given to infected and noninfected mice, and fluorescence intensity was measured subsequently in serum. RV
increased transit time in infant mice. Increased motility was detected at 24 h postinfection (h p.i.) and persisted up to 72 h p.i in
pups. Both loperamide and atropine decreased intestinal motility and attenuated diarrhea. Analysis of passage of fluorescent
dextran from the intestine into serum indicated unaffected intestinal permeability at the onset of diarrhea (24 to 48 h p.i.). We
show that RV-induced diarrhea is associated with increased intestinal motility via an activation of the myenteric nerve plexus,
which in turn stimulates muscarinic receptors on intestinal smooth muscles.
IMPORTANCE
We show that RV-infected mice have increased intestinal motility at the onset of diarrhea, and that this is not associated with
increased intestinal permeability. These new observations will contribute to a better understanding of the mechanisms involved
in RV diarrhea.
W
hile rotavirus (RV) is well established as a major cause of
severe acute gastroenteritis in young children all over the
world, the mechanisms behind diarrhea and vomiting are still
largely unknown. The fluid loss due to diarrhea may be caused by
several mechanisms (1, 2). High concentrations of poorly absorbable compounds may create an osmotic force causing a loss of
fluid across the intestinal epithelium (osmotic diarrhea). Secretory diarrhea is characterized by an overstimulation of the intestinal secretory capacity not coupled to an inhibition of fluid absorptive mechanisms. Furthermore, if the barrier function of the
epithelium is compromised by loss of epithelial cells or disruption
of the tight junction, hydrostatic pressure in blood and lymphatic
vessels may cause water and electrolytes to accumulate in lumen
(exudative diarrhea) (1). In most types of diarrhea, more than one
of these pathophysiological mechanisms are involved.
Several hypotheses have been forwarded to explain RV-induced diarrhea, including a functional role of the virus-encoded
enterotoxin NSP4 (3–5), malabsorption secondary to failing
transport of electrolytes and/or glucose/amino acids (6), villus
ischemia (7–10), and stimulation of the enteric nervous system
(ENS) (11) and of enterochromaffin (EC) cells (12). At the cellular
and tissue levels, a new understanding of the disease mechanisms
is beginning to emerge (2). Intestinal permeability and motility
are the two physiological mechanisms most rarely investigated,
but currently they are being implicated in diarrheal syndromes (2,
13). Intestinal permeability has only rarely been investigated in
humans infected with RV (14). In the early 1980s, Stintzing and
coworkers (15) assessed the intestinal permeability in young children with RV infection using polyethylene glycols (PEG) of different molecular weights, and a significantly low urinary recovery
March 2014 Volume 88 Number 6
of PEG was noted. A similar observation was made in adults by
Serrander and coworkers (16). Moreover, Johansen et al. (17)
found that children with acute RV infection excreted less PEG in
their urine than healthy children and children with enteropathogenic Escherichia coli (EPEC) infections. In vitro studies, on the
other hand, have shown that RV can increase the permeability of
polarized human epithelial Caco-2 cells (18), probably due to a
reorganization of the tight junction proteins claudin-1, occludin,
and ZO-1 (19). Furthermore, the RV enterotoxin NSP4 induces
paracellular leakage in polarized epithelial cells and prevents lateral targeting of ZO-1 (20). In line with this, it has also been demonstrated in Ussing chamber experiments that the electrical tissue
conductance is increased in RV-infected intestines (11, 21).
Intestinal motility has only occasionally been studied during
RV infection. It is well established that stimulating the vagal efferent nerves to the gut increases intestinal motility via a direct effect
on the intestinal musculature and/or via an excitation of the intestinal cells of Cayal (22). Atropine, the classical muscarinic receptor antagonist, can block these effects. It is also well known that
Received 5 October 2013 Accepted 20 December 2013
Published ahead of print 26 December 2013
Editor: S. López
Address correspondence to Lennart Svensson, [email protected].
C. I. and M. H. contributed equally to this work.
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
doi:10.1128/JVI.02927-13
Journal of Virology
p. 3161–3169
jvi.asm.org
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Grupo de Virologia, Unidade de Microbiologia Médica, Centro de Malária e Outras Doenças Trópicais, Instituto de Higiene e Medicina Tropical, Universidade Nova de
Lisboa, Lisbon, Portugala; Division of Molecular Virology, Department of Clinical and Experimental Medicine, University of Linköping, Linköping, Swedenb; Division of
Medical Microbiology, Department of Clinical and Experimental Medicine, University of Linköping, Linköping, Swedenc
Istrate et al.
MATERIALS AND METHODS
Animals. RV-naive BALB/c, 8-week-old adults and 5- to 7-day-old infant
mice (B&K Laboratories, Sollentuna, Sweden) were used. They were
housed in standard cages with free access to food and water. Pregnant
females were transferred to individual cages 1 week before the expected
day of birth, and offspring remained with their mother during the experimental period. The animal ethics committee in Stockholm approved the
experimental protocol (N291/010, N289/09).
Rotavirus infection and criteria for diarrhea. Mice (adults and infants) were orally infected with 10 ␮l/animal (100⫻ the dose causing
diarrhea in 50% of animals [DD50]) of wild-type murine RV (EDIM
strain) as previously described (28). Only infant mice were examined for
diarrhea, since adult mice have previously been shown not to develop
diarrhea (29). Adult mice were infected to investigate whether any
changes in motility and permeability occur in the absence of diarrhea.
Diarrhea was defined as a liquid yellow stool revealed by gentle abdominal
palpation as described elsewhere (11, 30). The percentage of mice with
diarrhea was calculated as the number of pups with typical diarrheal stools
(loose, yellow, liquid feces) divided by the total number of pups in the
same group. Mice were confirmed to have diarrhea before being included
in the treatment studies with loperamide and atropine.
Motility studies. In the motility experiments, adult (n ⫽ 6 to 13 at
each time point) and infant (n ⫽ 5 to 13 at each time point) BALB/c mice
received orally 10 ␮l of 4-kDa FITC-dextran probe (FD-4; Sigma) at 6, 12,
24, 48, 72, and 96 h p.i. The probe was also given to noninfected mice. The
adult mouse dose was 0.5 mg/mouse, and the infant mouse dose was 0.25
mg/mouse. FITC-dextran was dissolved in Milli-Q water, and the solutions were freshly prepared before each experiment. For time points of 24
h p.i. and later, diarrhea was confirmed in all infected infant mice that
received the probe. After 30 min for adults and 15 min for infant mice, the
animals were sacrificed and the whole intestines, from stomach to rectum,
were used for determination of transit time by UV light measurement by
taking photos using an FITC-specific filter (filter 520DF30; Bio-Rad) in a
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ChemiDoc system (ChemiDoc XRS; Bio-Rad). The front part of the main
accumulating FITC-dextran was defined from the photo, and the software
program Illustrator CS6 was used to more exactly measure the intestinal
length and migration of the FITC-dextran probe.
Intestinal transit was calculated at different time points on how far the
FITC-dextran probe has passed as a percentage of the entire length of the
intestine, from the pylorus to the rectum. Motility experiments were performed on two different occasions.
Antidiarrheal drug experiments. Loperamide hydrochloride and atropine sulfate salt monohydrate were purchased from Sigma (loperamide,
no. 34014; atropine, no. A0257) and dissolved in Milli-Q water. Loperamide (10 mg/kg) or atropine (6 mg/kg) was administered orally to adult
and infant mice at 40 and 44 h p.i. Four hours after the second dose, at 48
h p.i., mice received 4-kDa FITC dextran and were then sacrificed after 15
(infant) and 30 (adult) min for determination of transit time. In infant
mice the antidiarrheal effect of loperamide and atropine was checked at 4
h after the first dose and 4 h after the second dose (48 h p.i.) of treatment,
a time point associated with severe diarrhea of almost all mice. In the
loperamide experiment, mice also were checked for diarrhea on the following day and received a final dose of loperamide at 64 h p.i., the effect of
which was investigated 4 h later.
Intestinal permeability studies. The transmural passage into the
blood of fluorescent 10- and 4-kDa dextran was used to assess the barrier
characteristics of infant and adult mice infected with RV from 6 up to 96
h p.i. compared to noninfected animals. The barrier properties were described in two ways: (i) by a permeability index equal to the ratio between
the relative uptake of fluorescent 10- and 4-kDa dextran and (ii) by the
fold increase in either marker compared to the basal value for noninfected
animals. The values are given as fluorescence intensity (FI). The relative
permeation of the 10- and 4-kDa probes was used to calculate a permeability index (R) through their relative fluorescence intensities, F10 and F4
in blood/serum, i.e., R ⫽ F10/F4.
Briefly, RV-naive BALB/c mice, noninfected and RV infected, received
orally a mixture of 4-kDa FITC-dextran (adult mouse dose of 2.5 mg/
mouse; infant mouse dose of 0.7 mg/mouse) and 10-kDa rhodaminedextran (adult dose, 5.0 mg/mouse; infant dose, 1.4 mg/mouse). The fluorescently conjugated dextran (4-kDa FITC-dextran; no. FD-4; SigmaAldrich) and R881 (10-kDa rhodamine-dextran [4-kDa FITC-dextran;
no. FD-4; and 10-kDa rhodamine-dextran; no R881; Sigma-Aldrich]) was
freshly prepared in Milli-Q water before use. Blood was collected in serum
tubes (BD Microtainer tubes; no. 365951) 3 h after tracer administration
and centrifuged for 5 min at 1,677 ⫻ g. The serum was diluted 1/50 in
Milli-Q water, and the fluorescence intensities of FITC (494/518 nm) and
rhodamine (565/580 nm) were measured with a fluorescence spectrophotometer (LS-3B; Perkin-Elmer, Waltham, MA).
Histology. Specimens (0.5 cm) were taken from the duodenum, the
middle part of the jejunum, and the lowest part of ileum from adult and
infant mice, noninfected and infected, at 48 h p.i. The specimens were
fixed in 4% phosphate-buffered formaldehyde (no. 02176; Histolab, Sweden) and then embedded in paraffin. From each specimen, sections of
5-␮m thickness were cut and placed onto glass slides (SuperFrost Plus
Menzel no. 06400; Histolab, Sweden). The paraffin was removed through
2 steps of 5-min incubations in tissue clear solution (code 1466; Sakura,
Tokyo, Japan), and the tissues were hydrated through a series of ethanol
(EtOH) baths (99.5% EtOH twice for 5 min each, 95% EtOH for 5 min,
70% EtOH for 5 min) followed by a final step in phosphate-buffered saline
(PBS). Slides were then stained with hematoxylin and eosin for 5 min,
rinsed in tap water, and mounted with Pertex mounting medium (no.
00814; Histolab, Sweden) and a cover glass.
Statistical analysis. Data were analyzed with nonparametric twotailed Mann-Whitney test and Kruskal-Wallis test using Prism Software
5.0 (GraphPad, San Diego, CA) or Fisher’s exact test. Values were considered significant at P ⱕ 0.05.
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naturally occurring opioids can be used as antidiarrheal agents,
but their adverse central nervous effects make them undesirable as
remedies. Loperamide, an opioid receptor agonist, differs from
other similar drugs by binding predominantly to intestinal tissue,
and when given orally, it gains almost no access to the central
nervous system (CNS). Its antidiarrheal action is thought to result
from diminished intestinal motility by interfering with the myenteric plexa (22, 23). Furthermore, loperamide can inhibit intestinal secretion caused by bacterial enterotoxins (24, 25). Regarding
RV infections, Yamashiro and coworkers (26) found a significant
effect of the stool score on days 3 to 5 of treatment. Meta-analyses
confirmed that patients treated with loperamide are less likely to
have diarrhea 24 h after treatment and present with a shorter
duration of diarrhea and fewer stools than patients in the placebo
group (27).
Previous human permeability and motility studies have been
performed at a time when children already have documented RV
diarrhea. Thus, it has remained unresolved whether the effects on
motility and permeability are early or late host responses following diarrhea rather than the actual cause of diarrhea onset.
The objective of this study was to investigate whether and how
intestinal permeability and motility are altered at the onset of RV
diarrhea. To address this issue, different-sized fluorescent dextran
doses were given to RV-infected and noninfected adult and infant
mice to determine both permeability and transit kinetics. Intestinal motility was also studied after administration of the opioid
receptor agonist loperamide or the muscarinic receptor antagonist atropine. Our study gives an important new understanding of
the mechanisms triggering diarrhea.
Rotavirus Permeability and Motility
RESULTS
Rotavirus increases intestinal motility in adult and infant mice.
Alterations in intestinal transit/motility have been associated with
several forms of gastrointestinal diseases, promoting the development of pharmaceutical drugs that attenuate intestinal motility.
For RV infection it still remains unclear whether RV could increase intestinal transit and thereby contribute to diarrhea.
Therefore, to obtain a global view of the possible involvement
of increased motility in RV diarrhea, we infected infant and adult
mice with murine RV at 10 ␮l/animal (100⫻ DD50 diarrhea doses)
and characterized the kinetics of intestinal transit of a 4-kDa
FITC-dextran probe. We observed that the probe passed along the
entire intestine within 15 min in infected infant mice at 48 h p.i.,
more rapidly than for noninfected mice (P ⱕ 0.001) (Fig. 1A and
B). Interestingly, the peak of transit at 48 h p.i. coincided with the
time of the most severe diarrhea symptoms in pups, as 12 out of 13
mice had diarrhea (Table 1). RV infection in mice was confirmed
by eosin and hematoxylin staining of tissue sections of different
parts of the small intestine (Fig. 2). The characteristic vacuolization of the epithelial cells was seen on villi but not in the crypts.
We next investigated whether adult mice, despite not responding with diarrhea following RV infection, have altered intestinal
motility. Theoretically, RV infection could stimulate the myenteric plexus and motility in the absence of diarrhea (2). Indeed, we
found that the infection in adult mice did not resulted in increased
intestinal motility, as determined by the transit of FITC-dextran
(Fig. 1C). However, a slight effect was found at 24 h p.i., a time
point typically associated with onset of diarrhea in pups.
March 2014 Volume 88 Number 6
Atropine, a muscarinic receptor antagonist, attenuates rotavirus motility and diarrhea. The motility studies with atropine
oral treatment, using a dose of 6 mg/kg, were performed as described in Materials and Methods. We observed a significant decrease of intestinal motility in infected infant and adult mice
treated with atropine compared to nontreated mice at 48 h p.i.
(Fig. 3A and B). In noninfected infant mice, atropine had a modest
effect on motility (Fig. 3A). Since the effect of atropine was also
observed in noninfected mice (Fig. 3A and B), it did not seem
likely to be virus specific.
We also investigated whether atropine could attenuate RVinduced diarrhea in infant mice. Only mice with confirmed diarrhea were included, and atropine was given at 40 and 44 p.i. The
TABLE 1 Opioid receptor agonist loperamide attenuates rotavirus
diarrhea
No. of mice suffering diarrhea at:
Infant mouse type (n)
40 h p.i. 44 h p.i. 48 h p.i. 64 h p.i. 68 h p.i.
EDIM infected,
untreated (13)
EDIM infected,
loperamide treated (9)
Noninfected, loperamide
treated (6)
Noninfected, untreated
(2)
12/13
12/13
12/13
12/13
12/13
8/9
0/8
0/8
6/8
2/8
0/6
0/6
0/6
0/6
0/6
0/2
0/2
0/2
0/2
0/2
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FIG 1 Intestinal motility is increased at the time point of severe diarrhea, 48 h p.i. (n ⫽ 5 to 13). (A) Illustration of the transit of fluorescent FITC-dextran in the
entire intestine of EDIM-infected and noninfected infant mice at 15 min after dextran treatment. FITC-dextran was visualized by a UV spectrophotometer
(Chemi-Doc with an FITC-specific filter; Bio-Rad) and was identified as a fluorescence-dependent white color reaching different intestinal segments. (B and C)
Transit of FITC-dextran in infant (B) and adult (C) mice 15 and 30 min after dextran treatment, respectively. Onset of diarrhea in infant mice occurred between
24 and 48 h p.i. and was most severe at 48 h p.i., with a yellow, loose form. Values are based on the transit length of FITC-dextran as a percentage of the total length
of the intestine. ***, P ⱕ 0.001 by Kruskal-Wallis test.
Istrate et al.
B) Jejunum at 48 h p.i. showing the characteristic vacuolization of cells in the middle and top of villi of a mouse with diarrhea and rotavirus in the stool. (C)
Noninfected tissue from the duodenum.
effect of the 2 doses of atropine was evaluated at 48 h p.i. In the
mock-treated group 6/7 still had diarrhea, in contrast to only 3/11
in the atropine-treated group (P ⬍ 0.05 by Fisher’s exact test)
(Table 2).
In summary, we conclude that the muscarinic receptor antagonist can attenuate the increased intestinal motility and reduce
diarrhea.
Stimulation of ␮-opioid receptors in the myenteric plexa attenuates rotavirus diarrhea and motility. To further address how
RV affects intestinal motility, we treated infected mice with loperamide, an opioid receptor agonist known for its effect of attenuating intestinal motility by interfering with the myenteric plexa
of the ENS. Infant and adult mice were treated with two doses of
loperamide (10 mg/kg, orally, 10 ␮l/mouse) at 40 and 44 h p.i. At
48 h p.i., mice were given 4-kDa FITC-dextran, and after 15 and 30
min in infant and adult mice, respectively, mice were sacrificed for
intestinal transit analysis (n ⫽ 5 to 13 in each group) (Fig. 4A to
FIG 3 Atropine decreases the intestinal motility in EDIM-infected infant and
adult mice at 48 h p.i. Transit of FITC-dextran in infant (A) and adult (B) mice
15 and 30 min after dextran treatment, respectively. FITC-dextran was visualized by a UV spectrophotometer (Chemi-Doc with an FITC-specific filter;
Bio-Rad) and was identified as a fluorescence-dependent white color reaching
different intestinal segments. Values are based on the transit length of FITCdextran as a percentage of the total length of the intestine. **, P ⱕ 0.01 by
Mann-Whitney test.
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C). We observed a decrease in intestinal motility in RV-infected
infant mice treated with loperamide compared to nontreated infected mice at 48 h p.i. (Fig. 4A and B). The FITC-dextran probe
was flushed from the untreated infected mice (Fig. 4A, line 4) but
was delayed in loperamide-treated and infected mice (Fig. 4A, line
5). Giving loperamide to noninfected infant mice had a modest
effect on motility (Fig. 4B). No significant effect on intestinal motility was observed in infected adult mice treated with loperamide
compared to infected nontreated adults at 48 h p.i. (Fig. 4C). The
drug again had only modest effect on noninfected adult mice (Fig.
4C). We conclude that opioid receptor agonists can attenuate the
increased intestinal motility in RV-infected mice.
We next investigated whether loperamide could attenuate RVinduced diarrhea using the same experimental protocol as that in
the transit studies, i.e., infant mice were given doses of loperamide
at 40 and 44 h p.i. In the mock-treated group, 12/13 had diarrhea
at 40 h p.i., which was maintained during the course of the experiment until termination at 68 h p.i. (Table 1), and among infected
mice 8/9 had diarrhea at 40 h p.i., before administration of loperamide (Table 1). Only the 8 mice with confirmed diarrhea were
included in the loperamide-treated group. After the first dose (40
h p.i.), none of them displayed diarrhea at 44 h p.i. (Table 1), and
they showed no sign of diarrhea following administration of a
second dose (48 h p.i.). The effect of loperamide was highly significant (P ⬍ 0.01 by Fisher’s exact test).
To study the pharmacological kinetics of loperamide, treated
mice were also inspected 20 h after administration of the second
dose (64 h p.i.), and surprisingly, 6/8 mice displayed diarrhea.
When all eight mice once again were given a single dose of loperamide, only 2/8 mice exhibited diarrhea 4 h later (Table 1) (68 h
p.i.), an effect that was statistically significant (P ⬍ 0.05 by Fisher’s
exact test). In summary, we conclude that the opioid receptor
TABLE 2 Muscarinic antagonist atropine attenuates rotavirus diarrhea
No. of mice suffering diarrhea at:
Infant mouse type (n)
40 h p.i.
44 h p.i.
48 h p.i.
EDIM infected, untreated (7)
EDIM infected, atropine treated (11)
7/7
11/13
7/7
2/11
6/7
3/11
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FIG 2 Histology of the small intestinal epithelium of EDIM-infected and noninfected infant mice. Tissue sections are stained with hematoxylin and eosin. (A and
Rotavirus Permeability and Motility
agonist loperamide can abolish diarrhea in a time-dependent
manner in this RV animal model.
The intestinal permeability to 4- and 10-kDa dextran probes
is unaffected at the onset (24 to 48 h p.i.) of rotavirus-induced
diarrhea in mice. The fluid loss in diarrhea most likely is multifactorial and may, in the case of RV infection, include disturbances in intestinal permeability. Previous human studies investigating intestinal barrier functions (15, 17) all were conducted at
a time point at which subjects already had diarrhea. Thus, it remains unclear whether permeability changes are a late host response following diarrhea and/or the result of extensive lesions of
the epithelia rather than the actual cause of diarrhea onset.
To address this issue, we orally administered 4-kDa FITC-dextran and 10-kDa rhodamine-dextran to infant and adult mice at 6,
12, 24, 48, 72, and 96 h p.i. Adult and infant animals were bled at
3 h after tracer administration to assess the transmural passage of
the probes. The use of two different-sized probes allowed us to
investigate in more detail whether the intestinal barrier was influenced by the RV infection. The plasma concentration of the two
probes, as reflected in their optical densities, was estimated during
the course of infection as a measure of their intestinal permeability. A ratio between the permeability of the two probes was also
determined (Table 3), as well as the change of permeability to
either compound (fold increase) compared to the corresponding
noninfected animal.
We found that infected infant, in contrast to adult, mice re-
March 2014 Volume 88 Number 6
TABLE 3 Permeability of dextran probes in rotavirus-infected infant
mice
10-kDa probe
4-kDa probe
Time point
(h p.i.)
Fold
increase FIa
Fold
increase FI
10-kDa/4-kDa
ratio
n
6
12
24
48
72
96
Noninfected
1.8
1.4
1.0
0.6
1.4
0.5
1.0
20.1
8.6
0.6
0.9
0.6
1.9
1.0
0.6
1.2
11.0
4.5
17.9
1.8
7.2
a
18.1 (0.9)
14.5 (1.3)
9.9 (2.1)
5.9 (0.4)
14.3 (1.2)
4.7 (1.0)
10.1 (0.3)
28.2 (8.6)
12.0 (2.6)
0.9 (0.2)
1.3 (0.3)
0.8 (0.0)
2.6 (0.9)
1.4 (0.4)
6
7
6
9
6
6
6
FI, mean fluorescence intensity. Data in parentheses are standard errors of the means.
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FIG 4 Loperamide decreases intestinal motility in EDIM-infected infant and
adult mice at 48 h p.i. (A) Transit of fluorescent FITC-dextran in the entire
intestine of EDIM-infected and noninfected and treated and nontreated infant
mice at 15 min after dextran treatment. FITC-dextran was visualized by a UV
spectrophotometer (Chemi-Doc with an FITC-specific filter; Bio-Rad) and
was identified as a fluorescence-dependent white color reaching different intestinal segments. (B and C) Transit of FITC-dextran in infant (B) and adult
(C) mice at 15 and 30 min after dextran treatment, respectively. Values are
based on the transit length of FITC-dextran as a percentage of the total length
of the intestine. P ⱕ 0.05 (*) and P ⱕ 0.01 (**) by Mann-Whitney test.
sponded with a significant increase in permeability for both the 4and 10-kDa probes at 6 and 12 h p.i. (P ⬍ 0.01; Mann-Whitney
test). The increase for the 4-kDa probe (Fig. 5A and B) was particularly dramatic. In contrast, at 24 h p.i. and up to 96 h p.i., the
permeability to the 4-kDa probe was attenuated compared to that
of noninfected mice, and a transient peak for the 10-kDa probe
was observed at 72 h p.i. The most important observation was that
no increased permeability was observed for the 4-kDa or the 10kDa probes at 24 or 48 h p.i. (Fig. 5A and B and Table 3), time
points when mice exhibited diarrhea, enterocyte vacuolization
(28), and villus atrophy in the jejunum part of the small intestine
(Fig. 2A and B). Intestinal lesions (vacuolization and atrophy)
were also observed in the duodenum and ileum, as well as apically
in the epithelium, but not in crypts. This suggests that these pathological changes were not sufficient to induce increased permeability for 4- and 10-kDa dextran probes.
A reduced surface area should principally affect the permeability of the 10- and 4-kDa probes to the same extent and yield a
parallel decline of either marker in the blood. However, if there is
a perturbation of the junctional integrity, the paracellular uptake
of the larger 10-kDa probe might be enhanced relative to the
smaller 4-kDa probe (termed the 10-kDa/4-kDa ratio) (31). In the
early phase of infection, the 10-kDa/4-kDa ratio was balanced,
based on a 20-fold increased for the 4-kDa probe and an 18-fold
increased for the 10-kDa probe. However, a pronounced 11-fold
increase in the 10-kDa/4-kDa ratio was observed at 24 h p.i and a
17.9-fold increase at 72 h p.i., caused by the significant tightening
of the epithelium for the 4-kDa probe (Table 3). Interestingly, at
48 h p.i., when the clinical signs of diarrhea are obvious, the 10kDa/4-kDa ratio was 4.5 and was caused by the decreased permeability of the 10-kDa probe (Table 3).
To get further insight into the mechanisms associated with the
onset of diarrhea, we investigated whether adult infected mice not
responding with diarrhea from the small intestine (28) had a perturbed intestinal barrier. Accordingly, a set of experiments identical to the infant mouse permeability studies was performed in
adult mice. The permeability of the 4-kDa probe was decreased at
time points 6, 12, and 24 h p.i. (Fig. 6A). A limited permeability
increase for the 4-kDa probe was observed at 48 and 72 h p.i. but
did not reach statistical significance. Rather, there was a significantly reduced permeability at 24 and 96 h p.i. (P ⬍ 0.05; MannWhitney test) (Fig. 6A). There were no clear changes observed in
the intestinal permeability of the 10-kDa probe (Fig. 6B) except at
12 h p.i. (P ⬍ 0.05; Mann-Whitney test). The 10-kDa/4-kDa ratio
was highest from 6 to 24 h p.i. and then at 96 h p.i. (Table 4). It is
Istrate et al.
FITC-dextran (A) and 10-kDa rhodamine-dextran (B) through the intestinal epithelium into serum at different time points p.i. was measured by spectrophotometry at different fluorophore wavelengths (494/518 and 565/580 nm). Values are given as fluorescence intensity (FI). P ⱕ 0.001 (***), P ⱕ 0.01 (**), and P ⱕ
0.05 (*) by Mann-Whitney test.
interesting that the increased ratio before 48 h p.i. seemed to be
due to reduction of permeability for the 4-kDa probe, while the
uptake of the 10-kDa probe remained virtually unaffected.
DISCUSSION
Several disease mechanisms, such as malabsorption, altered permeability, motility disturbances, stimulation of enterochromaffin
cells, activation of the ENS, and the RV NSP4 toxin, have been
associated with fluid loss in RV diarrhea (2, 3, 5–8, 12, 13). Some
of these mechanisms may indeed occur after the onset of diarrhea
rather than being the actual cause. In this study, we have investigated how intestinal transit (motility) and epithelial permeability
are related to the onset of RV diarrhea. These two important disease-associated mechanisms have not been studied together in the
early phase of RV diarrhea.
Motility of the small intestine, as in all parts of the digestive
tract, is controlled predominantly by excitatory and inhibitory
signals from the ENS; however, they are modulated by inputs from
the CNS, whereas intestinal secretion is controlled mainly by intrinsic nerves and in particular by the submucosal plexus (22). The
autonomic nervous system control of motility is exerted mainly
via the release of acetylcholine and the effect on muscarinic receptors. Hence, it is possible to block this motor response by atropine,
as also demonstrated in this study. Considering in vivo studies in
several experimental models of acute diarrhea, atropine has failed
to attenuate the rate of fluid secretion in the small intestine.
In the present study, gut motility was determined by studying
the intestinal transit of the 4-kDa FITC-dextran probe in infant
and adult mice. Interestingly, in infant mice, the onset of diarrhea
occurred concomitantly with a high intestinal transit of FITCdextran at 48 h p.i. (Fig. 1A and B). To get further insight into the
pathophysiological mechanisms responsible for the increased mo-
FIG 6 Intestinal permeability is not increased at the most critical time point of EDIM infection in adult mice. Permeability of 4-kDa FITC-dextran (A) and
10-kDa rhodamine-dextran (B) through the intestinal epithelium into serum at different times p.i. was measured by fluorescence at wavelengths specific for each
fluorophore (494/518 and 565/580 nm). Values are given as fluorescence intensity (FI). *, P ⱕ 0.05 by Mann-Whitney test.
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FIG 5 Intestinal permeability is not increased at the time for onset of diarrhea or at the time of the most severe diarrhea in infant mice. Permeability of 4-kDa
Rotavirus Permeability and Motility
TABLE 4 Permeability of dextran probes in rotavirus-infected adult
mice
4-kDa probe
Fold
increase
FIa
Fold
increase
FI
10-kDa/4kDa ratio
n
6
12
24
48
72
96
Noninfected
1.1
1.3
0.9
1.0
1.1
1.1
1.0
8.7 (0.3)
9.9 (0.4)
6.7 (0.6)
7.7 (0.2)
8.6 (0.2)
8.2 (0.4)
7.7 (0.6)
0.4
0.4
0.3
1.2
2.6
0.3
1.0
1.1 (0.2)
1.2 (0.2)
1.0 (0.1)
3.7 (2.5)
7.9 (3.6)
0.8 (0.0)
3.0 (1.0)
7.9
8.3
6.7
2.1
1.1
10.3
2.6
5
5
10
5
5
5
7
a
FI, mean fluorescence intensity. Data in parentheses are standard errors of the means.
tility, we treated noninfected and RV-infected mice with the
␮-opioid receptor agonist loperamide, attenuating the activity of
the myenteric plexus (23), and with atropine, blocking muscarinic
receptors on smooth muscles. Loperamide differs from other opioid analogues, as it binds predominantly to intestinal tissue. Thus,
when given orally it has virtually no access to the CNS. Loperamide is a widely used antidiarrheal agent, its effects being attributed to an inhibitory action on smooth muscle tone and peristalsis, attenuating both cholinergic and noncholinergic influences on
intestinal smooth muscles (32, 33). The effect of loperamide was
not restricted to infant mice, as adult noninfected and infected
mice responded with attenuated intestinal motility, strongly suggesting that the effect is not virus specific. The efficacy was very
pronounced, since the diarrhea in infant mice was completely
abolished in 8/8 mice 4 h after a single dose. When giving a second
dose, it appeared that the effect was time dependent, since 6/8
mice exhibited diarrhea 20 h after the second dose. In humans,
maximal plasma loperamide concentration is achieved within 3 to
5 h in humans (34), which corresponds to the significant effect
observed within 4 h in the mouse model. It has also been proposed
that loperamide, apart from its inhibitory action on smooth muscle tone, also can stimulate absorption of fluid, electrolytes, and
glucose; moreover, it can reverse the prostaglandin E2 (PGE2) and
cholera toxin-induced secretion to absorption (24). In previous
studies, loperamide has been administered to individuals with ongoing diarrhea; therefore, the question of whether increased gut
motility is associated with the onset of diarrhea or a systemic host
response following diarrhea is still unresolved.
The effect of atropine on infected infant mice after 2 doses (6
mg/kg) at 40 and 44 h p.i. determined a clear reduction in motility
as well as reduced diarrhea, where only 3/11 mice had diarrhea
after atropine treatment compared to 6/7 in the mock-treated
group.
In conclusion, our results obtained with atropine, the classical
muscarinic receptor antagonist, and loperamide, a ␮-opioid receptor agonist, clearly suggest that ENS is involved in the motility
response to rotavirus infection. We found earlier that the fluid loss
in RV diarrhea is at least partly induced by activation of the ENS
(11).
Adult mice were explored to determine whether motility
changes were associated with water and electrolyte loss (i.e., diarrhea) from the small intestine or whether motility changes occurred independently of diarrhea. Previous studies have shown
that the small intestine of infant mice (11, 35) but not adult mice
(28) respond with electrolyte and water secretion (diarrhea) fol-
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10-kDa probe
Time point
(h p.i.)
lowing infection with murine RV. In diarrhea-resistant adult mice
there was a most modest increase of intestinal motility at 24 h p.i.
(Fig. 1C) but no overall increased motility during the course of
infection.
The participation of prostaglandins (PGs) in RV diarrhea may
be another factor explaining the different time courses for secretion and motility. It has been shown that PGs are produced under
the influence of microorganisms and have immune-modulatory,
anti-inflammatory, as well as proinflammatory actions (36).
Moreover, they can stimulate water secretion (37–40), an effect
that can be blocked by drugs attenuating nerve activity, such as
hexamethonium, a nicotinic receptor blocker, or lidocaine, a local
anesthetic (41), indicating that nerves are involved. Loperamide is
also a potent inhibitor of intestinal fluid secretion induced by
PGE2 and cholera toxin. In children with RV gastroenteritis, elevated levels of PGE2 and PGF2 in both plasma and stools have been
found, and treatment with the COX inhibitor aspirin reduced the
duration of diarrhea (26). Moreover, intravenous infusion of PG
causes abdominal cramps (42), inhibits intestinal motility (43,
44), and stimulates water secretion (24, 38, 45). In addition, RV
induces COX2 mRNA and secretion of PGE2 already within 8 h
p.i. (46). These observations make it tempting to speculate that the
decreased motility with remaining diarrhea 72 to 96 h p.i. is induced by increasing concentrations of PGs. The action of PGs on
smooth muscles, whether they act directly on the muscle cells or
modulate of the release of neurotransmitters (47), is not yet fully
understood.
In several in vitro animal and human studies, both increased
and decreased permeability have been reported. Regarding RV,
only a few human studies have been done (15–17); all of these
studies were conducted on children already infected with RV, and
the time course for conceivable permeability changes was not
studied.
The use of a combination of two markers of different sizes to
measure changes in the small intestinal permeability has proved to
be a sensitive and specific approach (16, 48). Interestingly, infected infant mice responded with a significant increase in permeability for both probes at 6 and 12 h p.i. (Fig. 5A and B), whereas
adults showed an increase only for the 10-kDa probe at 12 h p.i.
(Fig. 6A and B). The increased permeability at 6 and 12 h p.i. was
due to the administration of the fecal debris but was not associated
with diarrhea or increased motility. Furthermore, oral administration of a 10% fecal suspension from uninfected animals did not
result in diarrhea. An obvious conclusion from this unexpected
observation is that increased permeability per se is not sufficient to
cause diarrhea, since at these early time points mice do not have
diarrhea. The onset of diarrhea (24 to 48 h p.i.) was not associated
with increased permeability to the two dextran probes despite
significant lytic lesions in the small intestine at 48 h p.i. (Fig. 2A
and B). This conclusion is further supported by our observation
that even adult mice have similar pathological lesions at the same
time points but no diarrhea (28). Apparently, RV-induced pathological lesions per se in the small intestine (Fig. 2) are not sufficient to account for permeability changes or electrolyte changes
during RV infections. A similar observation was reported by Heyman and coworkers (49), who studied transepithelial fluxes of
horseradish peroxidase (HRP; molecular mass of about 44 kDa)
from mucosa to serosa in EDIM-infected mice in an Ussing chamber. During RV diarrhea (days 2 to 8), no change in mucosal-toserosal permeability to HRP was observed. On the other hand,
Istrate et al.
13.
14.
15.
16.
17.
18.
ACKNOWLEDGMENT
This work was supported by Swedish Research Council (LS) 320301.
19.
REFERENCES
1. Field M. 2003. Intestinal ion transport and the pathophysiology of diarrhea. J. Clin. Investig. 111:931–943. http://dx.doi.org/10.1172/JCI18326.
2. Hagbom M, Sharma S, Lundgren O, Svensson L. 2012. Towards a
human rotavirus disease model. Curr. Opin. Virol. 2:408 – 418. http://dx
.doi.org/10.1016/j.coviro.2012.05.006.
3. Estes MK 2003. The rotavirus NSP4 enterotoxin: current status and challenges, p 207–224. In Desselberger U, Gray J (ed), Viral gastroenteritis.
Elsevier, Amsterdam, the Netherlands.
4. Dong Y, Zeng CQ, Ball JM, Estes MK, Morris AP. 1997. The rotavirus
enterotoxin NSP4 mobilizes intracellular calcium in human intestinal
cells by stimulating phospholipase C-mediated inositol 1,4,5trisphosphate production. Proc. Natl. Acad. Sci. U. S. A. 94:3960 –3965.
http://dx.doi.org/10.1073/pnas.94.8.3960.
5. Ball JM, Tian P, Zeng CQ, Morris AP, Estes MK. 1996. Age-dependent
diarrhea induced by a rotaviral nonstructural glycoprotein. Science 272:
101–104. http://dx.doi.org/10.1126/science.272.5258.101.
6. Lundgren O, Svensson L. 2001. Pathogenesis of rotavirus diarrhea. Microbes
Infect. 3:1145–1156. http://dx.doi.org/10.1016/S1286-4579(01)01475-7.
7. Lundgren O, Svensson L. 2003. The enteric nervous system and infectious diarrhea, p 51– 67. In Desselberger U, Gray J (ed), Viral gastroenteritis. Elsevier, Amsterdam, the Netherlands.
8. Ramig RF. 2004. Pathogenesis of intestinal and systemic rotavirus infection. J. Virol. 78:10213–10220. http://dx.doi.org/10.1128/JVI.78.19.10213
-10220.2004.
9. Osborne MP, Haddon SJ, Spencer AJ, Collins J, Starkey WG, Wallis TS,
Clarke GJ, Worton KJ, Candy DC, Stephen J. 1988. An electron microscopic investigation of time-related changes in the intestine of neonatal
mice infected with murine rotavirus. J. Pediatr. Gastroenterol. Nutr.
7:236 –248. http://dx.doi.org/10.1097/00005176-198803000-00014.
10. Osborne MP, Haddon SJ, Worton KJ, Spencer AJ, Starkey WG, Thornber D, Stephen J. 1991. Rotavirus-induced changes in the microcirculation of intestinal villi of neonatal mice in relation to the induction and
persistence of diarrhea. J. Pediatr. Gastroenterol. Nutr. 12:111–120. http:
//dx.doi.org/10.1097/00005176-199101000-00021.
11. Lundgren O, Peregrin AT, Persson K, Kordasti S, Uhnoo I, Svensson L.
2000. Role of the enteric nervous system in the fluid and electrolyte secretion of rotavirus diarrhea. Science 287:491– 495. http://dx.doi.org/10
.1126/science.287.5452.491.
12. Hagbom M, Istrate C, Engblom D, Karlsson T, Rodriguez-Diaz J, Buesa
J, Taylor JA, Loitto VM, Magnusson KE, Ahlman H, Lundgren O,
3168
jvi.asm.org
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
Svensson L. 2011. Rotavirus stimulates release of serotonin (5-HT) from
human enterochromaffin cells and activates brain structures involved in
nausea and vomiting. PLoS Pathog. 7:e1002115. http://dx.doi.org/10.1371
/journal.ppat.1002115.
Michelangeli F, Ruiz MC 2003. Physiology and pathophysiology of the
gut in relation to viral diarrhea, p 23–50. In Desselberger U, Gray J (ed),
Viral gastroenteritis. Elsevier, Amsterdam, the Netherlands.
Kukuruzovic R, Robins-Browne RM, Anstey NM, Brewster DR. 2002.
Enteric pathogens, intestinal permeability and nitric oxide production in
acute gastroenteritis. Pediatr. Infect. Dis. J. 21:730 –739. http://dx.doi.org
/10.1097/00006454-200208000-00007.
Stintzing G, Johansen K, Magnusson KE, Svensson L, Sundqvist T.
1986. Intestinal permeability in small children during and after rotavirus
diarrhoea assessed with different-size polyethylene glycols (PEG 400 and
PEG 1000). Acta Paediatr. Scand. 75:1005–1009. http://dx.doi.org/10
.1111/j.1651-2227.1986.tb10331.x.
Serrander R, Magnusson KE, Sundqvist T. 1984. Acute infections with
Giardia lamblia and rotavirus decrease intestinal permeability to lowmolecular weight polyethylene glycols (PEG 400). Scand. J. Infect. Dis.
16:339 –344. http://dx.doi.org/10.3109/00365548409073958.
Johansen K, Stintzing G, Magnusson KE, Sundqvist T, Jalil F, Murtaza
A, Khan SR, Lindblad BS, Mollby R, Orusild E, Svensson L. 1989.
Intestinal permeability assessed with polyethylene glycols in children with
diarrhea due to rotavirus and common bacterial pathogens in a developing community. J. Pediatr. Gastroenterol. Nutr. 9:307–313. http://dx.doi
.org/10.1097/00005176-198910000-00008.
Svensson L, Finlay BB, Bass D, von Bonsdorff CH, Greenberg HB. 1991.
Symmetric infection of rotavirus on polarized human intestinal epithelial
(Caco-2) cells. J. Virol. 65:4190 – 4197.
Dickman KG, Hempson SJ, Anderson J, Lippe S, Zhao L, Burakoff R,
Shaw RD. 2000. Rotavirus alters paracellular permeability and energy
metabolism in Caco-2 cells. Am. J. Physiol. Gastrointest. Liver Physiol.
279:G757–G766.
Tafazoli F, Zeng CQ, Estes MK, Magnusson KE, Svensson L. 2001.
NSP4 enterotoxin of rotavirus induces paracellular leakage in polarized
epithelial cells. J. Virol. 75:1540 –1546. http://dx.doi.org/10.1128/JVI.75.3
.1540-1546.2001.
Isolauri E, Kaila M, Arvola T, Majamaa H, Rantala I, Virtanen E,
Arvilommi H. 1993. Diet during rotavirus enteritis affects jejunal permeability to macromolecules in suckling rats. Pediatr. Res. 33:548 –553. http:
//dx.doi.org/10.1203/00006450-199306000-00002.
Furness J (ed). 2006. The enteric nervous system. Blackwell Publishing,
Inc., Oxford, United Kingdom.
Ooms LA, Degryse AD, Janssen PA. 1984. Mechanisms of action of
loperamide. Scand. J. Gastroenterol. Suppl. 96:145–155.
Sandhu BK, Tripp JH, Candy DC, Harries JT. 1981. Loperamide: studies
on its mechanism of action. Gut 22:658 – 662. http://dx.doi.org/10.1136
/gut.22.8.658.
Watt J, Candy DC, Gregory B, Tripp JH, Harries JT. 1982. Loperamide
modifies Escherichia coli, heat-stable enterotoxin-induced intestinal secretion. J. Pediatr. Gastroenterol. Nutr. 1:583–586. http://dx.doi.org/10
.1097/00005176-198212000-00023.
Yamashiro Y, Shimizu T, Oguchi S, Sato M. 1989. Prostaglandins in the
plasma and stool of children with rotavirus gastroenteritis. J. Pediatr. Gastroenterol. Nutr. 9:322–327. http://dx.doi.org/10.1097/00005176-198910000
-00010.
Li ST, Grossman DC, Cummings P. 2007. Loperamide therapy for acute
diarrhea in children: systematic review and meta-analysis. PLoS Med.
4:e98. http://dx.doi.org/10.1371/journal.pmed.0040098.
Kordasti S, Istrate C, Banasaz M, Rottenberg M, Sjovall H, Lundgren O,
Svensson L. 2006. Rotavirus infection is not associated with small intestinal fluid secretion in the adult mouse. J. Virol. 80:11355–11361. http://dx
.doi.org/10.1128/JVI.00152-06.
Ward RL, McNeal MM, Sheridan JF. 1990. Development of an adult
mouse model for studies on protection against rotavirus. J. Virol. 64:
5070 –5075.
Kordasti S, Sjovall H, Lundgren O, Svensson L. 2004. Serotonin and
vasoactive intestinal peptide antagonists attenuate rotavirus diarrhoea.
Gut 53:952–957. http://dx.doi.org/10.1136/gut.2003.033563.
Menzies I. 1984. Transmucosal passage of inert molecules in health and
disease, p 527–543. In Skadhauge E, Heintze K (ed), Intestinal absorption
and secretion. MTP Press, Boston, MA.
Van Nueten JM, Janssen PA, Fontaine J. 1974. Loperamide (R 18 553),
Journal of Virology
Downloaded from http://jvi.asm.org/ on April 8, 2014 by LINKOPINGS UNVERSITSBIBLIOTEK
during convalescence (days 9 to 14), when virus was no longer
present in the mucosa, there was a 10-fold increase in HRP transport. They concluded that RV causes a transient rise in the gut
permeability during convalescence (49). In agreement with this
observation, we found a significant transient rise in permeability
72 h p.i. in both infant (P ⬍ 0.01; Mann-Whitney test) and adult
mice.
In infant mice, the 10-kDa/4-kDa ratio was decreased early in
infection, at 6 and 12 h p.i., and at 48 h p.i. This reduced ratio at the
time of diarrhea was apparently due to a reduction of the penetration of the 10-kDa probe. A most interesting observation was that
the 4-kDa probe had reduced uptake from 24 to 72 h p.i in infant
mice compared to uninfected infant mice. A similar observation
was obtained for the 10-kDa probe at 24 to 48 h p.i in infant mice.
These characteristics likely reflect both the surface area absorption
and the barrier properties of the different probes sizes as described
by the 10-kDa/4-kDa ratio.
In conclusion, we show that RV-infected mice have increased
intestinal motility at the onset of diarrhea, and this is not associated with any increased intestinal permeability. These new observations may contribute to a better understanding of the mechanisms involved in RV diarrhea.
Rotavirus Permeability and Motility
33.
34.
36.
37.
38.
39.
40.
41.
March 2014 Volume 88 Number 6
42.
43.
44.
45.
46.
47.
48.
49.
ing the small intestinal fluid secretion caused by arachidonic acid, prostaglandin E1 and prostaglandin E2 in the rat in vivo. Acta Physiol. Scand. 130:633–
642. http://dx.doi.org/10.1111/j.1748-1716.1987.tb08186.x.
Bergstrom S, Duner H, von Pernow EUB, Sjovall J. 1959. Observations
on the effects of infusion of prostaglandin E in man. Acta Physiol. Scand.
45:145–151. http://dx.doi.org/10.1111/j.1748-1716.1959.tb01686.x.
Thor P, Konturek JW, Konturek SJ, Anderson JH. 1985. Role of prostaglandins in control of intestinal motility. Am. J. Physiol. 248(Part 1):
G353–G359.
Bennett A, Eley KG, Scholes GB. 1968. Effect of prostaglandins E1 and E2
on intestinal motility in the guinea-pig and rat. Br. J. Pharmacol. 34:639 –
647. http://dx.doi.org/10.1111/j.1476-5381.1968.tb08493.x.
Karim SM, Adaikan PG. 1977. The effect of loperamide on prostaglandin
induced diarrhoea in rat and man. Prostaglandins 13:321–331. http://dx
.doi.org/10.1016/0090-6980(77)90011-9.
Rossen JW, Bouma J, Raatgeep RH, Buller HA, Einerhand AW. 2004.
Inhibition of cyclooxygenase activity reduces rotavirus infection at a postbinding step. J. Virol. 78:9721–9730. http://dx.doi.org/10.1128/JVI.78.18
.9721-9730.2004.
Wennmalm A. 1978. Prostaglandin-mediated inhibition of noradrenaline release. II. Dual mechanism behind its frequency-dependence. Acta
Physiol. Scand. 102:199 –204.
Weaver LT, Chapman PD, Madeley CR, Laker MF, Nelson R. 1985.
Intestinal permeability changes and excretion of micro-organisms in
stools of infants with diarrhoea and vomiting. Arch. Dis. Child. 60:326 –
332. http://dx.doi.org/10.1136/adc.60.4.326.
Heyman M, Corthier G, Petit A, Meslin JC, Moreau C, Desjeux JF. 1987.
Intestinal absorption of macromolecules during viral enteritis: an experimental study on rotavirus-infected conventional and germ-free mice. Pediatr. Res.
22:72–78. http://dx.doi.org/10.1203/00006450-198707000-00017.
jvi.asm.org 3169
Downloaded from http://jvi.asm.org/ on April 8, 2014 by LINKOPINGS UNVERSITSBIBLIOTEK
35.
a novel type of antidiarrheal agent. Part 3. In vitro studies on the peristaltic
reflex and other experiments on isolated tissues. Arzneimittelforschung
24:1641–1645.
Nakayama S, Yamasato T, Mizutani M. 1977. Effects of loperamide on
the gastric and intestinal motilities in the guinea pig and dog. Nihon
Heikatsukin Gakkai Zasshi 13:45–53. http://dx.doi.org/10.1540/jsmr1965
.13.45.
Killinger JM, Weintraub HS, Fuller BL. 1979. Human pharmacokinetics
and comparative bioavailability of loperamide hydrochloride. J. Clin.
Pharmacol. 19:211–218. http://dx.doi.org/10.1002/j.1552-4604.1979
.tb01654.x.
Wolf JL, Cukor G, Blacklow NR, Dambrauskas R, Trier JS. 1981.
Susceptibility of mice to rotavirus infection: effects of age and administration of corticosteroids. Infect. Immun. 33:565–574.
Scher JU, Pillinger MH. 2009. The anti-inflammatory effects of prostaglandins. J. Investig. Med. 57:703–708. http://dx.doi.org/10.231/JIM
.0b013e31819aaa76.
Sandhu JS, Fraser DR. 1983. Effect of dietary cereals on intestinal permeability in experimental enteropathy in rats. Gut 24:825– 830. http://dx
.doi.org/10.1136/gut.24.9.825.
Finck AD, Katz RL. 1972. Prevention of cholera-induced intestinal secretion in the cat by aspirin. Nature 238:273–274. http://dx.doi.org/10.1038
/238273a0.
Jacoby HI, Marshall CH. 1972. Antagonism of cholera enterotoxin by
anti-inflammatory agents in the rat. Nature 235:163–165. http://dx.doi
.org/10.1038/235163a0.
Vane JR. 1971. Inhibition of prostaglandin synthesis as a mechanism of
action for aspirin-like drugs. Nat. New Biol. 231:232–235. http://dx.doi
.org/10.1038/newbio231232a0.
Brunsson I, Sjoqvist A, Jodal M, Lundgren O. 1987. Mechanisms underly-