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Infection and Immunity, November 2001, p. 7173-7177, Vol. 69, No. 11
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.11.7173-7177.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
A 4.1-Kilodalton Polypeptide in the Cultural
Supernatant of Mycoplasma fermentans Is One of the
Substances Responsible for Induction of Interleukin-6 Production by
Human Gingival Fibroblasts
Akira
Hasebe,*
Ken-Ichiro
Shibata, and
Tsuguo
Watanabe
Department of Oral Pathobiological Science,
Graduate School of Dental Medicine, Hokkaido University, Nishi 7, Kita 13, Kita-ku, Sapporo 060-8586, Japan
Received 3 April 2001/Returned for modification 20 June
2001/Accepted 20 August 2001
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ABSTRACT |
The cultural supernatant of Mycoplasma fermentans
induced interleukin-6 production by human gingival fibroblasts. The
active entities were divided into hydrophilic and hydrophobic
substances. In this study, we purified a 4.1-kilodalton polypeptide
from the hydrophilic substances. It reacted with polyclonal antibodies to M. fermentans and activated human macrophages.
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TEXT |
Most bacterial modulins, which are
involved in inflammatory responses, are cell wall components. However,
mycoplasmas, wall-less microbes, stimulate lymphocytes, natural killer
cells, and monocytes/macrophages to produce cytokines and
chemokines (17). The cytokine production-inducing activity
exists mostly in fractions containing hydrophobic compounds such as
lipoproteins or lipoglycans (17). One
macrophage-stimulating lipopeptide with an approximate
molecular mass of 2 kDa has been extracted and isolated from
Mycoplasma fermentans cells and is named
macrophage-activating lipopeptide 2 (MALP-2) (16).
It has been reported that MALP-2 induces production of interleukin-6 (IL-6), IL-8, IL-10, tumor necrosis factor alpha (TNF-
), monocyte chemoattractant protein 1, and macrophage inflammatory
protein-1 by human monocytes (11). M. fermentans and M. salivarium induce production of
IL-6 and IL-8 by human gingival fibroblasts (HGF) (6, 23)
and cell surface expression of intercellular adhesion molecule 1 in HGF
(3). The activity of M. salivarium exists in cell membrane and intracellular fractions, and a water-soluble active entity (20.6-kDa protein) has been partially purified from intracellular fractions (6). Except for this,
water-soluble substances in mycoplasmas capable of activating HGF have
not been identified.
In this study, we found that cultural supernatants of M. fermentans induced cytokine production by HGF and we tried to
purify and characterize the active entities.
Organisms and culture conditions. M.
fermentans
ATCC 19989 was grown in PPLO broth (Difco
Laboratories, Detroit, Mich.) supplemented with 2% (vol/vol) horse
serum (GIBCO Life Technologies, Inc., Grand Island, N.Y.), 1% (wt/vol)
yeast extract (Difco), 1% (wt/vol) D-glucose, 0.002%
(wt/vol) phenol red, 0.05% (wt/vol) thallium acetate, and penicillin G
(1,000 U/ml). Cultures were incubated at 37°C for 48 h, at which
point the growth was in the mid-logarithmic phase, and centrifuged at 100,000 × g for 1 h to separate the cultural
supernatant, which was used for purification of the active entities.
Cell cultures.
HGF prepared and used in a previous study
(3) were cultured in Dulbecco's modified Eagle's (DME)
medium (GIBCO) containing 10% (vol/vol) fetal bovine serum (FBS),
penicillin G (100 U/ml), and streptomycin (100 µg/ml) in plastic
culture dishes with a medium change every 3 days for 7 to 10 days until
the cells reached confluency. The single-cell suspension prepared by
trypsin treatment was seeded in wells of a flat-bottom microplate.
After incubation for 2 days at 37°C in an atmosphere of 5%
CO2, the monolayers were washed three times with
DME medium without FBS [DME(
) medium], followed by the addition of
a test stimulant in 220 µl of DME() medium. Further incubation was
done at 37°C for 6 h. The culture plate was then centrifuged at
400 × g for 10 min.
THP-1 (a myelomonocytic cell line, JCRB 0112.1) cells obtained
from the Health Science Research Resources Bank (Osaka, Japan) were cultured in RPMI 1640 medium (GIBCO) containing 10%
(vol/vol) FBS, penicillin G (100 U/ml), and streptomycin (100 µg/ml)
in a plastic culture bottle. The cells were collected by centrifugation at 400 × g for 10 min, washed three times with RPMI
1640 medium without FBS [RPMI() medium], and suspended in RPMI()
medium at a cell concentration of 5 × 106/ml. A 200-µl volume of the cell suspension
was transferred to round-bottom wells of a microculture plate, and a
test stimulant in 20 µl of RPMI() medium was then added to the
wells. Further incubation was done at 37°C for 6 h.
Assay for cytokines.
The concentrations of IL-6, TNF-, and
IL-1
in the cultural supernatant were determined by using TiterZyme
enzyme-linked immunosorbent assay kits (PerSeptive Diagnostics
Inc., Cambridge, Mass.) in accordance with the manufacturer's
instructions. The detection limits of the enzyme-linked immunosorbent
assay kit are 10.9 pg/ml for IL-6, 28.1 pg/ml for TNF-, and
2.69 pg/ml for IL-1.
Ability of M. fermentans cultural supernatants
to induce IL-6 production by HGF.
M. fermentans
was grown in medium identical to that described above. A 2-ml aliquot
of the culture was taken periodically and divided into two portions.
One portion was used to determine the pH and the number of viable
cells by using liquid and agar media described by Hayflick
(7). The other portion was centrifuged at 100,000 × g for 1 h to separate the cultural supernatant, which was used to determine IL-6 production-inducing activity.
Determination of amounts of proteins, amino groups, and
carbohydrates.
The amount of proteins was determined by the method
of Dully and Grieve (4), the amount of compounds with
amino groups was determined by the colorimetric ninhydrin
method using L-arginine as a standard (25),
and the amount of carbohydrates was determined by the
phenol-sulfuric acid method (5).
SDS-PAGE.
Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis (SDS-PAGE) was performed with 16%
polyacrylamide gels by the method of Laemmli (13).
Proteins were stained by using a silver stain plus kit (Bio-Rad
Laboratories, Hercules, Calif.). Kaleidoscope polypeptide standards
(Bio-Rad) were used to estimate molecular weights.
Enzyme treatment.
Proteinase K was purchased from
Takara Biomedicals (Otsu, Japan), and lipoprotein lipase (EC
3.1.1.34.) was purchased from Sigma-Aldrich Co. (St. Louis, Mo.). A
0.02:1 (wt/wt) reaction mixture of enzyme and the sample
separated from the cultural supernatant of M. fermentans was treated at 37°C for 2 h and then tested for IL-6 production-inducing activity.
Antiserum.
Japanese White rabbits were injected subcutaneously
with 2 ml of a 1:1 (vol/vol) mixture of Freund's incomplete adjuvant
(Difco) and a cell suspension (500 µg of protein/ml) of
M. fermentans grown in PPLO broth (Difco) supplemented
with 2% (vol/vol) rabbit serum (GIBCO) on day 0, subcutaneously with
the same volume of the same mixture on day 14, and intraperitoneally
with 1 ml of the cell suspension on day 21. Sera were drawn before
immunization (preimmune serum) and 1 week after the final immunization
(anti-M. fermentans serum).
IL-6 production-inducing activity in cultural supernatants of
M. fermentans.
Almost in parallel with viable
counts, the IL-6 production-inducing activity increased with
prolongation of the incubation time up to 150 h, at which
point the activity had reached a maximum (Fig.
1). Then, the activity decreased
gradually, to approximately 80% of the maximum at 220 h, and
remained at that level for up to 380 h. Activity was not detected
in medium not inoculated with the organism.
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FIG. 1.
Growth of M. fermentans in liquid
medium and IL-6 production-inducing activity in the cultural
supernatant. M. fermentans was grown in liquid
medium, and a 2-ml aliquot of the culture was taken periodically and
divided into two portions. One portion was used to determine the pH
( ) and the number of viable cells (CFU/ml,  ). The other was
centrifuged at 100,000 × g for 1 h to
separate the cultural supernatant, which was used to determine the
ability to induce IL-6 production by HGF ( ).
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Purification of active entities.
Proteins in the cultural
supernatant were precipitated with ammonium sulfate between 0 and 30%,
30 and 60%, and 60 and 100% saturation and were examined for
activity. Most of the activity (97%) was recovered in the proteins
precipitating with ammonium sulfate between 30 and 60% saturation
(P60, Table 1). In order to characterize
the active entities in P60, the effect of enzymes on the activity was
investigated. The activity was reduced to 30% by treatment with
proteinase K and lipoprotein lipase (Table 2), suggesting that proteins and
lipoproteins are involved in expression of the activity.
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TABLE 1.
IL-6 production-inducing activity in proteins separated
from supernatants of M. fermentans cultures by ammonium
sulfate
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P60 was fractionated by reversed-phase high-performance liquid
chromatography (HPLC) with a preparative Nucleosil 120-7 C
18 column (10 by 300 mm; Chemco Scientific Co.
Ltd., Osaka, Japan).
Fractionation was carried out by using the
following program:
at time zero, 5%
N,
N'-dimethylformamide (DMF)-95% milli-Q water
(MQW); at 15 min, 5% DMF-95% MQW; at 60 min, 5% DMF-95%
2-propanol;
and at the end, 5% DMF-95% 2-propanol. Each fraction was
dried
in vacuo at 45°C, dissolved in MQW, and examined for IL-6
production-inducing
activity. The activity was recovered in fractions
with two peaks
eluted at hydrophilic and hydrophobic regions. The
hydrophobic
fractions seemed to contain lipoproteins or lipopeptides
that
had already been characterized (
16). However,
hydrophilic substances
in
M. fermentans capable of
inducing production of cytokines have
not been identified. Therefore,
we were very much interested in
the active entities in the hydrophilic
fractions, peak A (Fig.
2).
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FIG. 2.
Protein profile (solid line) obtained by reversed-phase
chromatography of P60 on an HPLC column. P60 was applied to a
preparative Nucleosil 120-7 C18 column (10 by 300 mm;
Chemco Scientific Co. Ltd.). Fractionation was carried out by using the
following program: at time zero, 5% DMF-95% MQW; at 15 min, 5%
DMF-95% MQW; at 60 min, 5% DMF-95% 2-propanol; and at the end, 5%
DMF-95% 2-propanol. The flow rate was 1.0 ml/min. Each fraction was
dried in vacuo at 45°C, dissolved in MQW, and examined for IL-6
production-inducing activity (). Abs, absorbance.
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The effect of polymyxin B on the IL-6 production-inducing activity of
peak A was investigated to eliminate the possibility
that the activity
was attributable to lipopolysaccharides that
might contaminate peak A. HGF were preincubated with polymyxin
B (0, 5,000, and 10,000 U/ml)
because polymyxin B is an antibiotic
that destroys the biological
activities of lipopolysaccharides
and then stimulated with peak A at
37°C for 6 h. The activity
was not affected by polymyxin B (data
not
shown).
Peak A was then fractionated by size-exclusion HPLC with an Asahipak
GS-520 column (7.6 by 500 mm; Asahi Chemical Industry
Co. Ltd., Tokyo,
Japan). The fractionation was done by elution
with MQW. Each fraction
was dried in vacuo at 45°C, dissolved
in MQW, and examined for IL-6
production-inducing activity. The
activity was recovered in fractions
with a major peak (peak
B).
Peak B was further fractionated by reversed-phase HPLC with an
ODP-50 column (4.6 by 250 mm; Shoko Co. Ltd., Tokyo, Japan).
Fractionation was carried out by using the following program:
at time
zero, 0.05% trifluoroacetic acid (TFA)-20% acetonitrile;
at 5 min, 0.05% TFA-20% acetonitrile; at 20 min, 0.05% TFA-80%
acetonitrile; and at the end, 0.05% TFA-80% acetonitrile. Each
fraction was dried in vacuo at 45°C, dissolved in MQW, and examined
for IL-6 production-inducing activity. The activity was recovered
in
fractions with a major peak (peak
C).
Properties of the active entities in peak C.
Peak C activated
macrophages to produce TNF- and IL-1 (data not shown).
Peak C was found to contain substances with amino groups and
carbohydrates. The specific activity of peak C calculated on the basis
of the amount of amino group-containing substances was about 75-fold
higher than that of P60, while the activity calculated on the basis of
the amount of carbohydrate-containing substances was lower than that of
P60 (Table 3).
The effect of lipoprotein lipase on the IL-6 production-inducing
activity of peak C was investigated. A 0.02:1 (wt/wt) reaction
mixture
of lipoprotein lipase and peak C was treated at 37°C for
2 h and
then tested for the activity. It was found that lipoprotein
lipase had
no effect on the activity of peak C (data not
shown).
SDS-PAGE of peak C revealed one dense band with a molecular mass of 4.1 kDa (Fig.
3). Thus, the active entity in
peak C was
found to be a 4.1-kDa polypeptide (P4.1).
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FIG. 3.
SDS-PAGE. SDS-PAGE of polypeptide standards (A; 4.0 µg
of protein) and peak C (B; 0.3 µg of protein) was performed with a
16% polyacrylamide gel. Proteins were stained by using a silver stain
plus kit (Bio-Rad).
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M. fermentans cells and peak C were spotted onto two
nitrocellulose membranes. To avoid nonspecific binding of antibodies,
each of the membranes was blocked with 5% skim milk in
phosphate-buffered
saline. One membrane was then reacted with
anti-
M. fermentans serum, and the other was reacted
with preimmune serum. Each of
antibodies was detected by horseradish
peroxidase-conjugated goat
anti-rabbit immunoglobulin G and a
DAB substrate (Vector Laboratories,
Inc., Burlingame, Calif.).
Anti-
M. fermentans serum, but not preimmune
serum,
reacted with
M. fermentans cells and peak C (Fig.
4). The
fact that only anti-
M.
fermentans serum reacted with peak C demonstrated
that P4.1 was
derived from
M. fermentans.
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FIG. 4.
Detection of substances reacted with anti-M.
fermentans serum. M. fermentans cells (A, 4 µg of protein; B, 0.4 µg of protein) and peak C (C, 0.06 µg of
protein) were spotted onto two nitrocellulose membranes. Each of the
membranes was blocked with 5% skim milk in phosphate-buffered saline.
One membrane was then reacted with anti-M.
fermentans serum, and the other was reacted with preimmune
serum. Each of antibodies was detected by horseradish
peroxidase-conjugated goat anti-rabbit immunoglobulin G and a
DAB substrate (Vector Laboratories, Inc.).
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Mycoplasmal lipoproteins activate lymphocytes,
monocytes/macrophages, and fibroblasts, and the activity
resides in the N-terminal
lipopeptide moieties (
16,
22).
Lipoproteins are not released
in a water-soluble form from intact
mycoplasma cells because they
are anchored to the cell membranes by the
N-terminal lipid moiety.
Therefore, we were interested in
mycoplasma-derived and hydrophilic
substances capable of activating
mammalian cells because we thought
that hydrophilic substances are
capable of interacting with target
cells more easily and play more
important pathological roles than
hydrophobic substances such as
lipoproteins. Recently, we have
partially purified and characterized
hydrophilic substances from
M. salivarium that are
responsible for induction of IL-6 production
by HGF (
6).
In this sense, we were very much interested in
the finding that the
cultural supernatant of
M. fermentans induced
IL-6
production by HGF. The activity existed in hydrophilic and
hydrophobic
substances in the cultural supernatant. Hydrophobic
substances are
possibly lipoproteins or lipopeptides liberated
in some way from cell
membranes of
M. fermentans and solubilized
in the
cultural supernatant, possibly by association with serum
albumin in
horse serum, an essential ingredient of growth medium.
The active
entity in hydrophilic substances was found to be a
4.1-kDa peptide,
named P4.1, with carbohydrates (Table
3). P4.1
is presumably a product
of
M. fermentans or, less likely, a fragment
separated
from proteins or lipoproteins in cell membranes of the
organism by some
enzymes, because the activity increased almost
in parallel with viable
counts in cultures and reached a maximum
at the end of the log phase
(Fig.
1).
P4.1 activates TNF-
production by macrophages. MALP-2 has
been purified from the cell membrane of
M. fermentans
(
16) and
is thought to be the N-terminal lipopeptide
moiety released from
lipoproteins degraded by some proteolytic enzyme.
Therefore, it
is very likely that the cultural supernatant of
M. fermentans contains lipopeptides such as MALP-2.
Lipopeptides are possibly
eluted at the hydrophobic region in
reversed-phase HPLC. However,
P4.1 was eluted at the hydrophilic region
and is resistant to
lipoprotein lipase. Therefore, it is speculated
that P4.1 is another
activator of
macrophages.
M. fermentans was first isolated (
19,
20)
from the human lower genital tract and is thought to be a common
inhabitant
of the genital tract.
M. fermentans has been
suggested to be associated
with rheumatoid arthritis (RA) and AIDS
because
M. fermentans was also isolated from RA
patients (
32), detected in synovial
fluid of RA patients
by PCR methods (
9,
21), and isolated
from AIDS patients
(
10,
14).
Large amounts of cytokines such as TNF-
, IL-1
, IL-6, and IL-8 are
produced in the inflamed rheumatoid synovial fluid of
RA patients and
play crucial roles in the pathophysiology of RA
(
28). The
finding that P4.1 and lipoproteins induce IL-6 production
by HGF
suggests that they are capable of inducing IL-6 production
by synovial
fibroblasts.
Many studies on AIDS have suggested that mycoplasmas, including
M. fermentans, are involved for some reasons as
possible cofactors
in AIDS pathogenesis. For example, higher titers of
antibodies
to these mycoplasmas have been detected in human
immunodeficiency
virus-infected patients than in noninfected
individuals (
30),
and
M. fermentans has
been shown to have the ability to invade
host cells (
15,
29) and induce TNF-
secretion by monocytes,
which is known to
activate human immunodeficiency virus replication
(
18).
The finding that P4.1 induces TNF-
production by macrophages
suggests that it may play some pathological role in
AIDS.
Periodontitis is an inflammatory disorder characterized by bone
resorption. IL-6 is an inflammatory cytokine that plays an
etiological
role in this disease. IL-6 alone does not induce bone
resorption by
osteoclast formation, but soluble IL-6 receptors
trigger the formation
in the presence of IL-6 (
12).
Porphyromonas gingivalis, a gram-negative, anaerobic bacterium, is thought to
be
one of the pathogens in periodontitis.
P. gingivalis
possesses
the ability to induce IL-6 production by HGF
(
27). Baker et
al. reported that IL-6 production induced
by infection with
P. gingivalis contributes to alveolar bone
loss (
1). P4.1 also
induces IL-6 production by HGF. In
addition,
M. fermentans has
been detected in human
saliva (
2,
24). This suggests that
P4.1 plays some
etiological role in
periodontitis.
Temporomandibular disorder is a disease in which pain and impaired
mandibular movement appear to arise directly from degenerative
or
inflammatory changes within the temporomandibular joint, but
its
precise pathogenesis has not been elucidated. It has been
suggested
that the development of temporomandibular disorder may
be traced to a
single traumatic event that occurred before the
manifestation of
symptoms of the disease (
26). However, we detected
M. fermentans in the synovial fluid of patients with
temporomandibular
disorder by PCR (
31). Recently, Henry et
al. also reported that
they detected some microbes, including
M. fermentans, in temporomandibular
joints
(
8). Therefore, P4.1 may play an etiological role in
temporomandibular disorder. For these reasons,
M. fermentans may
be involved in the pathogenicity of some oral
diseases.
This study has demonstrated that
M. fermentans produces
a hydrophilic polypeptide capable of inducing the production of
inflammatory
cytokines other than
lipoproteins.
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ACKNOWLEDGMENTS |
This work was partially supported by Grants-in-Aid for Scientific
Research (C) (10671762), which were provided by the Ministry of
Education, Culture, Sports, Science and Technology, Japan.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Oral Pathobiological Science, Graduate School of Dental Medicine,
Hokkaido University, Nishi 7, Kita 13, Kita-ku, Sapporo 060-8586, Japan. Phone: 81-11-706 4242. Fax: 81-11-706 4901. E-mail:
akkun{at}den.hokudai.ac.jp.
Editor:
R. N. Moore
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REFERENCES |
| 1.
|
Baker, P. J.,
M. Dixon,
R. T. Evans,
L. Dufour,
E. Johnson, and D. C. Roopenian.
1999.
CD4+ T cells and the proinflammatory cytokines gamma interferon and interleukin-6 contribute to alveolar bone loss in mice.
Infect. Immun.
67:2804-2809[Abstract/Free Full Text].
|
| 2.
|
Chingbingyong, M. I., and C. V. Hughes.
1996.
Detection of Mycoplasma fermentans in human saliva with a polymerase chain reaction based assay.
Arch. Oral Biol.
41:311-314[CrossRef][Medline].
|
| 3.
|
Dong, L.,
K.-I. Shibata,
Y. Sawa,
A. Hasebe,
Y. Yamaoka,
S. Yoshida, and T. Watanabe.
1999.
Transcriptional activation of mRNA of intercellular adhesion molecule-1 and induction of its cell surface expression in normal human gingival fibroblasts by Mycoplasma salivarium and Mycoplasma fermentans.
Infect. Immun.
67:3061-3065[Abstract/Free Full Text].
|
| 4.
|
Dully, J. R., and P. A. Grieve.
1975.
A simple technique for eliminating interference by detergents in the Lowry method of protein determination.
Anal. Biochem.
64:136-141[CrossRef][Medline].
|
| 5.
|
Fox, J. D., and J. F. Robyt.
1991.
Miniaturization of three carbohydrate analyses using a microsample plate reader.
Anal. Biochem.
195:93-96[CrossRef][Medline].
|
| 6.
|
Hasebe, A.,
K.-I. Shibata,
H. Domon,
L. Dong, and T. Watanabe.
1999.
Partial purification and characterization of the active entity responsible for inducing interleukin-6 production by human gingival fibroblasts from Mycoplasma salivarium cells.
Microbiol. Immunol.
43:1003-1008[Medline].
|
| 7.
|
Hayflick, L.
1965.
Tissue cultures and mycoplasmas.
Tex. Rep. Biol. Med.
23(Suppl. 1):285-303.
|
| 8.
|
Henry, C. H.,
C. V. Hughes,
H. C. Gerard,
A. P. Hudson, and L. M. Wolford.
2000.
Reactive arthritis: preliminary microbiologic analysis of the human temporomandibular joint.
J. Oral Maxillofac. Surg.
58:1137-1142[CrossRef][Medline].
|
| 9.
|
Johnson, S.,
D. Sidebottom,
F. Bruckner, and D. Collins.
2000.
Identification of Mycoplasma fermentans in synovial fluid samples from arthritis patients with inflammatory disease.
J. Clin. Microbiol.
38:90-93[Abstract/Free Full Text].
|
| 10.
|
Katseni, V. L.,
C. B. Gilroy,
B. K. Ryait,
K. Ariyoshi,
P. D. Bieniasz, and D. Taylor-Robinson.
1993.
Mycoplasma fermentans in individuals seropositive and seronegative for HIV-1.
Lancet
341:271-273[CrossRef][Medline].
|
| 11.
|
Kaufmann, A.,
P. F. Mühlradt,
D. Gemsa, and H. Sprenger.
1999.
Induction of cytokines and chemokines in human monocytes by Mycoplasma fermentans-derived lipoprotein MALP-2.
Infect. Immun.
67:6303-6308[Abstract/Free Full Text].
|
| 12.
|
Kotake, S.,
K. Sato,
K. J. Kim,
N. Takahashi,
N. Udagawa,
I. Nakamura,
A. Yamaguchi,
T. Kishimoto,
T. Suda, and S. Kashiwazaki.
1996.
Interleukin-6 and soluble interleukin-6 receptors in the synovial fluids from rheumatoid arthritis patients are responsible for osteoclast-like cell formation.
J. Bone Miner. Res.
11:88-95[Medline].
|
| 13.
|
Laemmli, U. K.
1970.
Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature
227:680-685[CrossRef][Medline].
|
| 14.
|
Lo, S. C.,
J. W. Shih,
P. B. Newton, 3rd,
D. M. Wong,
M. M. Hayes,
J. R. Benish,
D. J. Wear, and R. Y. Wang.
1989.
Virus-like infectious agent (VLIA) is a novel pathogenic mycoplasma: Mycoplasma incognitus.
Am. J. Trop. Med. Hyg.
41:586-600.
|
| 15.
|
Lo, S. C.,
M. M. Hayes,
R. Y. Wang,
P. F. Pierce,
H. Kotani, and J. W. Shih.
1991.
Newly discovered mycoplasma isolated from patients infected with HIV.
Lancet
338:1415-1418[CrossRef][Medline].
|
| 16.
|
Mühlradt, P. F.,
M. Kie,
H. Meyer,
R. Sümuth, and G. Jung.
1997.
Isolation, structure elucidation, and synthesis of a macrophage stimulatory lipopeptide from Mycoplasma fermentans acting at picomolar concentration.
J. Exp. Med.
185:1951-1958[Abstract/Free Full Text].
|
| 17.
|
Razin, S.,
D. Yogev, and Y. Naot.
1998.
Molecular biology and pathogenicity of mycoplasmas.
Microbiol. Mol. Biol. Rev.
62:1138-1144.
|
| 18.
|
Rosenberg, Z. F., and A. S. Fauci.
1989.
Induction of expression of HIV in latently or chronically infected cells.
AIDS Res. Hum. Retrovir.
5:1-4[Medline].
|
| 19.
|
Ruiter, M., and H. M. M. Wentholt.
1950.
Isolation of a pleuropneumonia-like organism in primary fusospirochetal gangrene of the penis.
J. Investig. Dermatol.
15:301-304[Medline].
|
| 20.
|
Ruiter, M., and H. M. M. Wentholt.
1953.
Isolation of a pleuropneumonia-like organism in a case of fusospirillary vulvovaginitis.
Acta Dermato-Venereol.
33:123-129[Medline].
|
| 21.
|
Schaeverbeke, T.,
C. B. Gilroy,
C. Bebear,
J. Dehais, and D. Taylor-Robinson.
1996.
Mycoplasma fermentans, but not M. penetrans, detected by PCR assays in synovium from patients with rheumatoid arthritis and other rheumatic disorders.
J. Clin. Pathol.
49:824-828[Abstract/Free Full Text].
|
| 22.
|
Shibata, K.-I.,
A. Hasebe,
T. Into,
M. Yamada, and T. Watanabe.
2000.
The N-terminal lipopeptide of a 44-kDa membrane-bound lipoprotein of Mycoplasma salivarium is responsible for the expression of intercellular adhesion molecule-1 on the cell surface of normal human gingival fibroblasts.
J. Immunol.
165:6538-6544[Abstract/Free Full Text].
|
| 23.
|
Shibata, K.-I.,
A. Hasebe,
T. Sasaki, and T. Watanabe.
1998.
Mycoplasma salivarium induces interleukin-6 and interleukin-8 in human gingival fibroblasts.
FEMS Immunol. Med. Microbiol.
19:275-283.
|
| 24.
|
Shibata, K.-I.,
M. Kaga,
M. Kudo,
L. Dong,
A. Hasebe,
H. Domon,
Y. Sato,
H. Oguchi, and T. Watanabe.
1999.
Detection of Mycoplasma fermentans in saliva sampled from infants, preschool and school children, adolescents and adults by a polymerase chain reaction-based assay.
Microbiol. Immunol.
43:521-525[Medline].
|
| 25.
|
Shibata, K.-I., and T. Watanabe.
1986.
Carboxypeptidase activity in human mycoplasmas.
J. Bacteriol.
168:1045-1047[Abstract/Free Full Text].
|
| 26.
|
Solberg, W. K.,
T. L. Hansson, and B. Nordströrm.
1985.
The temporomandibular joint in young adults at autopsy: a morphologic classification and evaluation.
J. Oral Rehabil.
12:303-321[Medline].
|
| 27.
|
Steffen, M. J.,
S. C. Holt, and J. L. Ebersole.
2000.
Porphyromonas gingivalis induction of mediator and cytokine secretion by human gingival fibroblasts.
Oral Microbiol. Immunol.
15:172-180[CrossRef][Medline].
|
| 28.
|
Suzuki, M.,
T. Tetsuka,
S. Yoshida,
N. Watanabe,
M. Kobayashi,
N. Matsui, and T. Okamoto.
2000.
The role of p38 mitogen-activated protein kinase in IL-6 and IL-8 production from the TNF-alpha- or IL-1 beta-stimulated rheumatoid synovial fibroblasts.
FEBS Lett.
465:23-27[CrossRef][Medline].
|
| 29.
|
Taylor-Robinson, D.,
H. A. Davies,
P. Sarathchandra, and P. M. Furr.
1991.
Intracellular location of mycoplasmas in cultured cells demonstrated by immunocytochemistry and electron microscopy.
Int. J. Exp. Pathol.
72:705-714[Medline].
|
| 30.
|
Wang, R. Y.,
J. W. Shih,
T. Grandinetti,
P. F. Pierce,
M. M. Hayes,
D. J. Wear,
H. J. Alter, and S. C. Lo.
1992.
High frequency of antibodies to Mycoplasma penetrans in HIV-infected patients.
Lancet
340:1312-1316[CrossRef][Medline].
|
| 31.
|
Watanabe, T.,
K.-I. Shibata,
T. Yoshikawa,
L. Dong,
A. Hasebe,
H. Domon,
T. Kobayashi, and Y. Totsuka.
1998.
Detection of Mycoplasma fermentans in synovial fluids of temporomandibular joints of patients with disorders in the joints.
FEMS Immunol. Med. Microbiol.
22:241-246[CrossRef][Medline].
|
| 32.
|
Williams, M. H.,
J. Brostoff, and I. M. Roitt.
1970.
Possible role of Mycoplasma fermentans in the pathogenesis of rheumatoid arthritis.
Lancet
ii:277-280[CrossRef].
|
Infection and Immunity, November 2001, p. 7173-7177, Vol. 69, No. 11
0019-9567/01/$04.00+0 DOI: 10.1128/IAI.69.11.7173-7177.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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