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A Study of Inflammatory Mediators in the Human Tympanosclerotic Middle Ear
Marie Forséni, MD, PhD;
Dan Bagger-Sjöbäck, MD, PhD;
Malou Hultcrantz, MD, PhD
Arch Otolaryngol Head Neck Surg. 2001;127:559-564.
ABSTRACT
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Objective To analyze immunocompetent cells as well as 2 factors involved in inflammation
and also thought to be involved in bone remodeling—interleukin 6 (IL-6)
and inducible nitric oxide synthase in the human middle ear, including the
tympanic membrane.
Design Biopsy specimens were obtained from the human middle ear and tympanic
membrane during surgery. Using an immunohistochemical technique, the expression
of macrophages, T cells, B cells, IL-6, and inducible nitric oxide synthase
were analyzed.
Materials Nine biopsy specimens from tympanic membranes in children having a transtympanic
ventilation tube inserted as a treatment for secretory otitis media and 11
biopsy specimens from tympanosclerotic plaques from patients with chronic
otitis media and tympanosclerosis.
Results More positively stained specimens showing macrophages, B cells, and
IL-6 were seen in the biopsy specimens from children with secretory otitis
media compared with the biopsy specimens from patients with chronic otitis
media and tympanosclerosis. The biopsy specimens from patients with chronic
otitis media and tympanosclerosis more often showed positive stainings for
inducible nitric oxide synthase than the biopsy specimens from children with
secretory otitis media. The presence of IL-6 and inducible nitric oxide synthase
was shown by staining to be mostly in the surface cells, while macrophages
and B cells were stained deeper in the tissues, in connective tissue, or around
sclerotic lesions.
Conclusions The 2 patient groups differed in antigen presentation so that macrophages,
B cells, and IL-6 were labeled more frequently in patients with secretory
otitis media, that is, an early phase of the disease. Inducible nitric oxide
synthase was seen more frequently in the patients with already established
tympanosclerosis in a later phase of the disease.
INTRODUCTION
TYMPANOSCLEROSIS (TS) is a condition that occurs in the middle ear,
including the tympanic membrane (TM), and presents itself as calcification
of connective tissue. The process is most often seen in the TM but may also
involve other sites in the middle ear.
Tympanosclerosis is seen as white chalky patches in the middle ear or
in the TM. It is, in the initial stages, seen as cheeselike masses of sclerotic
material and in the later stages as a harder, more bonelike material. Tympanosclerosis
has been well described by many investigators.1, 2, 3, 4, 5, 6, 7, 8
The TS plaques are located in the lamina propria of the TM. The microstructure,
as seen under the electron microscope, is composed of an irregular 3-dimensional
collagen lattice, enclosing spherical mineralized aggregates that are masses
of calcium phosphate. The calcification process resembles that occurring in
other diseases such as arteriosclerosis.7 The
patients suffering from TS usually have a history of either acute or chronic
otitis media.1, 4 When the TM is
solely engaged, the disease is usually defined as myringosclerosis (MS). The
TM is also the most common localization of the process. Myringosclerosis often
occurs in patients who underwent tympanoplasty and had ventilation tubes inserted.6 Tympanosclerosis can occur after a mild inflammatory
process as well as after severe repeated bouts of inflammatory disease. The
incidence of TS in previously analyzed materials from patients with otitis
media varies between 20% and 43%.1, 4, 6
Some patients develop hearing loss when the middle ear ossicles are more or
less fixed as a result of the TS process. The inner ear can also be affected
in severe cases in which the otic capsule is involved. This may lead to sensorineural
hearing loss and even deafness in rare cases. Depending on the localization,
there are different implications for the choice of treatment modalities (eg,
surgical procedures). When performing surgery, there is always a risk of creating
iatrogenic sensorineural hearing loss. The potential benefit of surgery in
these patients is questionable, according to Wielinga and Kerr.9
Myringosclerosis, by itself, seldom causes any hearing impairment; however,
when the TS is located in the epitympanum, it is common that the malleus head
and the body of the incus are fixed. In such a case it may be surgically difficult
to free them from the tympanosclerotic plaques. It is not uncommon to see
fixation of the stapes footplate to the walls of the oval window, and sometimes
even the tendon of the stapes can be calcified. When the tympanosclerotic
plaques are located on the promontory, the stapes may have to be mobilized
to make a space for the surgical procedure that, of course, is risky because
the inner ear can be traumatized. Sometimes, the plaques form where earlier
bone destruction of the promontory has occurred. In view of this, there is
a risk of penetration into the labyrinth when surgery is performed. When the
plaques are located near the facial nerve, special precaution is needed. There
is no definite curative treatment except surgery. Recurrences are frequent,
often resulting in poor hearing.
A successful model for producing acute otitis media10
and TS8, 11 in the rat has been
developed in Sweden. Our hypothesis is that there might be a factor(s) triggering
an immunological reaction sequence that eventually leads to formation of calcified
plaques. It would be useful to identify these factors to predict who will
be at risk for developing TS, as well as to prevent or even treat the disease.
In previous animal studies, the inflammatory response in the rat middle ear
has been analyzed using polyclonal and monoclonal protein-specific and cell-specific
antibodies.8, 12, 13
To our knowledge,this inflammatory response has not been analyzed in human
subjects. This study was performed to investigate the presence of macrophages,
T cells, and B cells in the human middle ear, including the TM. These cell
types are commonly seen in inflammatory response. Antibodies against these
cells were used in the study as well as antibodies against inducible nitric
oxide synthase (iNOS) and interleukin 6 (IL-6). Inducible nitric oxide synthase
is an enzyme, expressed in activated macrophages and known to produce nitric
oxide (NO) that causes vasodilation and kills pathogens. Interleukin 6 is
a cytokine that acts as a mediator between different immunocompetent cells.
It is also thought to participate in the maturation of cells into osteoclasts,14 which is a cell involved in bone repair. This study
compares the presentation of the aforementioned cells and factors in 2 different
patient groups: one group with secretory otitis media (SOM), a condition that
is known to have a high propensity (expressed as a percentage) to develop
into TS, and one group with already established TS.
PATIENTS, MATERIALS, AND METHODS
PATIENTS
We studied 2 patient groups from whom biopsy specimens were obtained.
Nine children (4 girls and 5 boys) with SOM who underwent the placement of
middle ear ventilating tubes were randomly selected. The children ranged in
age between 2 and 9 years (Table 1). All patients showed intact TMs with the appearance of clear effusion behind
them. No signs of purulent otitis media was present. Despite the appearance,
1 middle ear was found to be aerated. The surgery was performed with the indication
of a hearing disability. A special instrument was used to obtain a biopsy
specimen, creating a small hole in the TM, where the ventilation tube was
placed. These specimens are hereafter referred to as the SOM biopsy specimens.
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Table 1. Results of 9 Biopsy Specimens Obtained From Children With
Secretory Otitis Media*
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Eleven specimens from defined TS plaques in the TM or the middle ear
from patients who underwent tympanoplasty were removed during surgery. Ten
of the patients were females and 1 was male. Age ranged from 8 to 55 years
(Table 2). All patients had dry
TM perforations prior to surgery. There no history of otorrhea for several
months in these patients, except for 1 case in which ongoing otorrhea was
present at the time of surgery. All patients had some kind of conductive hearing
loss. The biopsy specimens were collected from patients being operated on
under general or local anesthesia. These specimens hereafter are referred
to as the TS biopsy specimens. The project was approved by the Swedish Ethical
Board (No. 92:40). All patients had been informed about the study and the
procedure before approving the obtaining of biopsy specimens. The information
was verbal and written. If the patient was younger than 18 years, the parents
were informed and gave their consent.
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Table 2. Results of 11 Biopsy Specimens Obtained From Patients Undergoing
Tympanoplasty and/or Ossiculoplasty*
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MATERIALS AND METHODS
All biopsy specimens were immediately fixed in 4% paraformaldehyde for
1 hour, then embedded in paraffin. They were subsequently serially sectioned
in 4-µm sections and put on glass slides. Thereafter, they were deparaffinized.
Blocking of endogenous peroxidase activity was performed by using 0.3% hydrogen
peroxide. The sections were preincubated with normal serum from the same animal
in which the secondary antibody was produced. The slides were then exposed
to the primary antibodies MAC 387, CD3, CD20, iNOS, and IL-6 (H-183) (Table 3).
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Table 3. Antibodies Used in This Study
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The biopsy specimens were incubated in darkness overnight. After rinsing,
the secondary antibody was applied. When the primary antibody was of monoclonal,
bioltinylated immunoglobulin-antimouse-IgG (Vectastain; Vector Laboratories,
Burlingame, Calif) was used. For polyclonal antibodies, biotinylated anti–rabbit
IgG was used (Scandinavian Diagnostic Services, Copenhagen, Denmark). Avidin-biotin
complexes (Vectastain) were added and the specimens were incubated with 3-amino-9-ethylcarbazole,
dimethylformamide, and 30% hydrogen peroxide, pH 5.0. The slides were kept
in darkness for 4 minutes, then rinsed with tap water. The sections were counterstained
with Mayer hemalun. Specimens in which the primary antibody had been replaced
by normal serum and phosphate-buffered solutions were used as negative controls.
In staining procedures including MAC 387, CD3, CD20, and iNOS, human tonsil
tissue, known to contain immunocompetent cells, was used as both a positive
and negative (Figure 1) control.
When staining for IL-6, rat lymph nodes were used as negative and positive
controls. The specimens were analyzed and photographed using a light microscope
(Axioplan; Carl Zeiss Ltd, Oberkochen, Germany). Since the biopsy specimens
varied in size and shape, it was difficult to compare the number of positive
cells in each section; therefore, the specimen was considered either positive
or negative for antibody staining. Data from the positive specimens were analyzed
using the Mann-Whitney test. The results are summarized in Table 1 and Table 2.
Statistical significance was set at P<.05. Table 3 lists the different antibodies
used in detail.
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Figure 1. A, Tonsil tissue used as a positive
control. Cells positive for antibodies against inducible nitric oxide synthase
are indicated by arrows. Bar indicates 200 µm. B, Tonsil tissue used
as a negative control, where no stainings could be seen. Bar indicates 400
µm.
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RESULTS
The number of positive specimens are presented as a percentage of the
whole group of biopsy specimens. Macrophages were frequently seen in this
study (MAC 387). When staining for MAC 387, all specimens (100%) in the SOM
group were positive (Figure 2).
The different mediators had various staining patterns that seemed to be equal
in the tissues of the 2 patient groups. Staining showed the presence of IL-6
(Figure 3) and iNOS mostly in mucosal
surface cells. Macrophages (Figure 4)
and B cells (CD20) were identified in the deeper cell layers, that is, deep
in the mucosa, in connective tissue, or around sclerotic lesions. The 2 different
patient groups had different staining patterns, that is, there was a different
frequency of positive specimens from the various stains. A higher number of
positively stained specimens was seen in the SOM biopsy specimens for IL-6
(8 of 9 specimens) (Figure 5), macrophages
(9 of 9 specimens) (Figure 6), and
B cells (7 of 9 specimens) compared with TS biopsy specimens, in which IL-6
was seen in 3 of 11 specimens, macrophages were seen in 5 of 11 specimens,
and B cells were seen in 1 of 11 specimens. The TS biopsy specimens more often
stained positively for iNOS (7 of 11 specimens) than did the SOM biopsy specimens
(1 of 9 specimens). There was only 1 biopsy specimen in the SOM group that
stained positively for iNOS. This specimen was obtained from an air-filled
middle ear. One biopsy specimen in the SOM group exhibited myringosclerosis.
The presence of T cells (CD3) was not seen in any of the specimens despite
several staining procedures. The tonsil tissues and lymph nodes used as positive
controls all showed distinct staining of the immunocompetent cells, while
the negative controls did not show any staining. Data from Table 1 and Table 2,
except CD3, which was not detected, were analyzed using the Mann-Whitney test.
There were statistically significant differences in MAC 387 (P = .006), CD20 (P = .004), and IL-6 (P = .003), while iNOS was not statistically significant
(P = .03).
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Figure 2. The different biopsy specimens
presented as a percentage of positive specimens in each study group. SOM indicates
biopsy specimens from patients having secretory otitis media; TS, biopsy specimens
from patients having tympanosclerosis; IL-6, interleukin 6; and iNOS, inducible
nitric oxide synthase.
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Figure 3. Staining against interleukin 6
in a biopsy specimen from the group with tympanosclerosis (the same specimen
as in Figure 4). The superficial cells (thin arrow) are stained as well as
the ciliae of the mucosa (thick arrows). Bar indicates 100 µm.
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Figure 4. Staining with MAC 387 in a biopsy
specimen from the group with tympanosclerosis (the same specimen as in Figure
3). The cells are stained deeply in the tissue (arrows). Bar indicates 200
µm.
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Figure 5. Staining against interleukin 6
in a biopsy specimen from the group with secretory otitis media (the same
specimen as in Figure 6). Positive cells are seen mostly in the surface (arrows).
Bar indicates 400 µm.
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Figure 6. Staining with MAC 387 in a biopsy
specimen from the group with secretory otitis media (the same specimen as
in Figure 5). Positive cells are seen deeply in the tissue (arrows). Bar indicates
400 µm.
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COMMENT
Biopsy specimens from 2 different patient groups have been investigated
in this study: 1 group with SOM, a condition that is known to have a high
propensity (expressed as a percentage) to develop into TS, and one group with
already established TS. To find out the differences in the presentation of
immunocompetent cells and some mediators, an immunohistochemical technique
was used. The presentation of macrophages, T cells, and B cells was analyzed
as well as 2 specific markers, IL-6 and iNOS, also thought to be involved
in bone repair.
Bacterial challange and release of IL-6 and IL-8 in humans was performed
by Larsson et al.15 In this study, epithelial
cells from a human lung carcinoma cell line and human alveolar macrophages
from healthy subjects were stimulated with different agents, among others
a strain of gram-positive bacteria. The dose-dependent release of IL-6 and
IL-8 was studied and found to be elevated. It showed that these cytokines
are produced by epithelial cells after stimulation by this group of bacteria
that are of the same type that often invade and colonize the middle ear in
acute and chronic otitis media.
Messenger RNA of inflammatory cytokines was studied by Hebda et al16 in rat middle ear mucosal tissue after challenge
with Streptococcus pneumoniae. The time-dependent
response was examined for the expression of selected cytokine genes. They
showed that the expression of IL-6 peaked at 24 to 48 hours after bacterial
inoculation. In our study, IL-6 expression was shown in a higher percentage
of SOM biopsy specimens compared with TS biopsy specimens. This supports the
finding that this particular cytokine peaks early and not late in the inflammatory
response.
Another mediator that was frequently seen in TS biopsy specimens in
this study of patients with chronic otitis media was iNOS. Inducible nitric
oxide synthase is 1 of 3 isoforms of this enzyme and is normally absent in
macrophages but expressed when activated by cytokines, leading to production
of NO, which can cause vasodilation and kill pathogens, for example, bacteria.
Nitric oxide is a radical molecule produced by iNOS and is produced in large
amounts once iNOS is formed.17 Nitric oxide
plays a role in the inflammatory response in the middle ear and also in the
formation of TS.
The bonelike material in the tympanosclerotic plaques can be thought
of as ectopic bone, produced in the TM and in the middle ear. Osteoblasts
produce NO, in response to cytokine production, according to a study by Hukkanen
et al.18 However, cytokine-induced NO release
reduced osteoclast activity. Macrophages were frequently seen in our study.
Since this kind of cell is thought to be a precursor to osteoclasts,14 there is a possibility of differentiation when a
signal is given. This could be, for example, a special cytokine or a combination
of several mediators.
Several components in the inflammatory response have been analyzed in
human middle ear biopsy specimens. The 2 patient groups differed somewhat
in antigen presentation. There were more positive specimens for macrophages,
B cells, and IL-6 in the SOM biopsy specimens. There were more positive specimens
for iNOS in TS biopsy specimens. There was only 1 specimen in the SOM group
that stained positively for iNOS. This patient was the only one who had an
air-filled middle ear cavity, while all of the others had fluid-filled middle
ear cavities. This is interesting because NO is a gas and obviously must have
been produced in the middle ear. If the production of NO was enhanced in this
specific patient or if it was inhibited in the others is difficult to speculate.
Further investigations are needed to distinguish significant differences between
the 2 patient groups.
Other differences between the antigen presentation were the location
in the tissue. This seemed to be similar in the 2 patient groups. The presence
of IL-6 and iNOS was most evident in the surface cells, such as in the mucosal
epithelium. Macrophages and B cells were located deep in the mucosa, in connective
tissue, or around sclerotic lesions. Three specimens in the TS group showed
negative results for all antibody stainings, indicating that the antigenic
expression was low. The calcified plaques have usually been present for a
long time, often for several years while the process cannot be regarded as
active. The SOM group presented a more active picture regarding the presence
of immunocompetent cells and mediators. Tympanosclerosis formation may start
at this early stage. T cells did not stain positive in any of the specimens.
This result prevailed in several trials. It is surprising that no T cells
were present in the biopsy specimens; our conclusion is that the inflammatory
process cannot be regarded as acute in any of our patients. The patients with
TS all had perforated TMs and chronic otitis media. Macrophages are likely
to be found in middle ear tissue from such patients, always being attacked
by antigens emanating from the environment. Inducible nitric oxide synthase
was frequently shown in these cells, apparently activated macrophages. The
superficial mucosal cell layer and the subepithelial tissues were stained
in the TS biopsy specimens. In 1 specimen, cilia from the middle ear mucosa
stained positive for IL-6 (Figure 1).
This staining pattern is in accord with the findings in another study (M.F.,
D.B.-S., M.H., Å. Melhus, MD, PhD, and A. F. Ryan, PhD, unpublished
data, 1999) in which rat middle ear mucosa was analyzed using antibodies against
IL-6. In both species, IL-6 seems to be produced by epithelial cells and immunocompetent
cells. In the SOM biopsy specimens obtained from patients with intact TMs
and no air in the middle ear, the labeling pattern was reversed, that is,
no iNOS was seen.
The TS plaques are seen as bonelike structures macroscopically and during
morphologic examination using a light and/or electron microscope. Therefore,
one might consider the TS plaque formation an ectopic production of bone.
Osteoclasts and osteoblasts produce a variety of cytokines including IL-6.19 Therefore, we were interested to analyze this cytokine
in our study. It has been shown that IL-6 stimulates bone resorption in vitro
and in vivo and that IL-6 induces osteoclastlike cell formation18
in human bone marrow cultures. Udagawa et al20
demonstrated that induction of osteoclast differentiation by IL-6 depends
on IL-6 receptor expression by osteoblasts, rather than osteoclast progenitors.
Pretreatment of co-cultures with dexamethasone was required for IL-6–dependent
formation of osteoclastlike multinucleated cells. In a study by Chole et al,21 osteoclastic resorption in the bullar bone in mongolian
gerbils was studied. The air-filled middle ear was pressurized to 10 mm Hg
above atmospheric pressure, which leads to increased osteoclastic resorption
of the inner surface of the bullar bone and bone formation on the outer surface.
This study shows that macrophages seem to be present in both patient groups
and, because they may differentiate into osteoclasts, this might happen when
they are stimulated by cytokines, among others, IL-6. The osteoblasts are
also induced by IL-6. This probably occurs early in the inflammatory process.
Interleukin 6 was expressed in the SOM biopsy specimens. Other inflammatory
mediators such as NO are probably involved and NO can be produced both by
activated macrophages and endothelial cells. Therefore, in a theoretical model,
these cells and their inflammatory mediators can interact in the human middle
ear, constituting some of the factors involved in a reaction sequence that
eventually leads to the formation of TS. It would be clinically useful if
a marker identifying patients with a higher risk for TS development could
be found, so that these patients could be clinically followed up and perhaps
also be treated. To prevent the calcification process, perhaps a stricter
indication for ventilation tube treatment could be used. Thus, children with
SOM could be treated with ventilation tubes only in one ear when needed. Another
possibility is prevention using drugs. Dexamethasone seems to exert an effect
on osteoblasts and osteoclasts and could be a useful tool. Fenspiride is another
drug that inhibits the development of MS by local administration.22 Many other options may be found in the future when
a more detailed map of early and late TS formation has been obtained.
CONCLUSIONS
More positive specimens were seen in the group with SOM regarding the
presence of macrophages, B cells, and IL-6 compared with the group with TS.
The TS biopsy specimens more often stained positively for iNOS than did the
SOM specimens. T cells could not be identified in any of the specimens. The
presence of IL-6 and iNOS was revealed by the antibody staining mostly in
the surface cells, such as the mucosal epithelium and the subepithelial tissues.
Macrophages and B cells were seen in deeper tissues, such as deep in the mucosa,
in connective tissue, or around sclerotic lesions.
AUTHOR INFORMATION
Accepted for publication February 21, 2001.
From the Departments of Otorhinolaryngology, Karolinska Institute and
Hospital, Stockholm, Sweden.
Corresponding author and reprints: Marie Forséni, MD, PhD,
Department of Plastic and Reconstructive Surgery, Karolinska Hospital, 171
76 Stockholm, Sweden (e-mail: marie.forseni{at}ood.ki.se)
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