Role of Streptococcus sanguis
and Traumatic Factors in
Emiko Isogai, DVM*
Hiroshi Isogai, DVM
Shigeaki Ohno, MD
Koichi Kimura, MD§
Keiji Oguma, MD¶
of Preventive Dentistry, Health Sciences University of Hokkaido, Hokkaido
of Experimental Animals, Sapporo Medical University, Sapporo
of Ophthalmology and Visual Sciences, Hokkaido University Graduate School
of Medicine, Hokkaido
of Biomedical Engineering, Hokkaido Institute of Technology, Sapporo
of Bacteriology, Okayama University, Graduate School of Medicine and
Dentistry, Okayama, Japan
KEY WORDS: Behçets disease, Streptococcus sanguis,
experimental model, germ-free mouse, ocular inflammation.
Background: The pathogenicity of Behçets disease
(BD) has been associated with the offending pathogen Streptococcus sanguis.
However, it is unclear that the bacterium is a true pathogen.
Materials and Methods: Germ-free
mice were inoculated with a clinical isolate (strain BD11320)
of S. sanguis. Mice received heat or mechanical stress on their oral
tissue before the bacterial infection. Colonization, immune responses
against the cell wall and synthetic peptides, and the cytokine profile
were examined. Uveitogenicity of the cell wall, lipoteichoic acid (LTA),
muramyl dipeptide (MDP), human hsp336351, S. sanguis-associated
peptides, and retina-associated peptides were examined.
Results: S. sanguis
colonized the oral cavity at 1058/mL saliva. The level of colonization
in mice given heat or mechanical stress was significantly higher than
the other groups. These mice showed typical oral ulcers after the bacterial
challenge and mild iridocyclitis. Skin lesions were spread whereas genital
ulcers were rare in these groups. Significant antibody production to
the selected peptides was observed in the experimental mice compared
with control animals. Inflammatory cytokines such as IL-2, IL-6, IFN-g,
and TNF-a were detected in oral tissue of the mice infected with S.
sanguis. Evidence suggests that the association with the cell wall or
with LTA can directly affect the degree of inflammation.
S. sanguis strain BD11320 is pathogenic for experimental
mice and can be a causative agent for BD. Molecular mimic peptides can
be implicated in the pathogenesis of BD. The cell wall of the bacteria
shows direct ocular inflammogenicity.
Behçets disease (BD) is a multisystemic disorder
presenting with recurrent oral and genital ulceration as well as uveitis,
often leading to blindness.1,2 The etiology and pathogenesis of this
syndrome remains obscure. We found that the proportion of S. sanguis
in the oral flora of patients with BD has significantly increased compared
with control subjects.3,4 Patients show hypersensitivity in skin tests
with the streptococcal antigens, and symptoms typical of BD are sometimes
provoked by an injection of the antigen.5 Recently, we showed antibody
crossreactivity from sera of patients with BD with synthetic peptides
that have homologies with proteins from S. sanguis.6
The concept of overexpression of hsp either on the
cell surface proper or as peptides presented by MHC products has been
central to the hypothesis that hsp-specific antibodies and T cells play
a role in the pathogenesis of human autoimmune disease.7 T cell response
to hsp60 and increased levels of hsp-60-specific antibodies in serum
have been found in patients with BD.8,9 These immune responses to hsp
have also been found in a number of human autoimmune diseases.7,9 Thus,
it is difficult to explain the pathogenesis of BD by hsp alone, even
if human hsp is homologous with hsp from S. sanguis.
Recently, we succeeded in the isolation and sequence determination
of the bes-1 encoding a streptococcal antigen that correlated with BD.10
The residues in a portion of the amino acid sequence show 60% similarity
to the human intraocular peptide Brn-3b. Brn-3, a subfamily of POU (pit-Oct
Unc) domain factors, contain three members, Brn3a, Brn3b, and Brn3c.11
POU domain proteins are a class of transcriptional regulators that appear
to have important roles in tissue-specific gene regulation. Pit1 plays
a critical role in the development of the pituitarity and regulation
of prolactin and growth hormone synthesis; Oct1 is an ubiquitous transcription
factor and Oct2 regulates immunoglobulin synthesis in B lymphocytes.
Brn-3b is first expressed in migrating, postmitotic ganglion cell precursors
in the ventricular zone of developing mouse retinas.12
Fox et al.13 noted that peptidoglycan provoked chronic
inflammation and retinal necrosis similar to that observed in eyes injected
with lipopolysaccharide. However, because of the crude nature of the
cell wall extracts, the specific basis for cell wall-induced inflammation
was not determined. It has been reported that Gram-positive cell walls
stimulate synthesis of tumor necrosis factor alpha and interleukin-6
by human monocytes.14 With regard to the intraocular inflammogenicity
of the cell wall, neither the metabolically inactive pathogens nor purified
sacculi caused significant reductions in retinal responsiveness, but
they evoked significant inflammation in both the posterior and anterior
segments of the eye.15
The aims of this study are to produce an experimental
model with mono-infection of S. sanguis by using germ-free mice. Under
the gnotobiotic condition, the effect of other microbiota can be eliminated.
We also examined whether the cell wall and its constituents are capable
of inciting significant intraocular inflammation because the determination
of the virulence factor is important.
MATERIALS AND METHODS
Bacterial Strain Used
S. sanguis strain BD113-20 was used for the experiments.
The strain was isolated from the oral cavity of a patient with BD3.
A similar serotype (so-called KTH-1) was found in more than half of
patients with BD, but not in healthy control subjects.4 Clinical isolates
belonging to serotype KTH-1 has been identified as S. oralis by their
biochemical and enzymatic properties.16 However, our isolates were different
from those strains in the analysis of DNA homology and cell wall sugar
constituents.17 Bacteria were cultured in BHI medium at 37C for
Preparation of the Cell
The S. sanguis cell wall was prepared by the method previously
described.18 LTA from S. sanguis and MDP were purchased from Sigma Chemical
Co. (St. Louis, MO). They were suspended in sterile Hanks balanced
salt solution (Sigma Chemical Co.) at a concentration of 1 mg/mL.
Germ-free IQI/Jic mice, bred from ICR mice, were
obtained from Japan Clea Co. Ltd. (Tokyo, Japan). Ninety-five female
and male mice were used at 4 to 5 weeks of age. Each infection group
consisted of 10 mice whereas the negative control groups consisted of
5. Mice were maintained at biohazard level 3. Food and drinking water
were autoclaved before use. Before and after the experiments, feces
and bedding were cultured on BHI agar with 7% horse blood under both
anaerobic and aerobic conditions. No bacteria were contaminating the
culture at any time. Specific pathogen-free ICR mice (males, 5 wk of
age) were used for the experiments to confirm uveitogenicity of various
bacterial components and synthetic peptides. These animals were fed
with standard laboratory chow and maintained in the standard light-dark
cycle. Animals were cared for in accordance with the ARVO Statement
for Use of Animals in Ophthalmic and Vision Research. The ethical committee
of the Health Sciences University of Hokkaido allowed the design of
Heat shock (HS) to induce severe inflammation was performed
on the left side of the tongue dorsum and buccal surface of germ-free
mice before S. sanguis infection. A spatula was preheated at 250˚C
in a heat-box (Inotech STDRI 250, Inotech Co. Ltd., Tokyo, Japan), cooled
to 180200˚C (the temperature was estimated as radial energy
by spot thermometer HT-7, Minorta Co. Ltd.), and then attached to the
surface of the oral tissues for 10 seconds under anesthesia by using
Nembutal (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan). The treated
area was 5 mm x 5 mm. The group undergoing heat-shock treatment was
designated as the HS group.
Mechanical Damage of the Mucosal Surface (Scraping: SCR)
This method was used as an induction of mild trauma on
the oral mucosal surface. The oral surface (the left side of the tongue
dorsum and buccal surface, 5 mm x 5 mm) was scraped by a dental end
excavator under anesthesia. The group with mechanical damage of the
mucosal surface was designated as the SCR group.
Mouse Colonization Experiments
In the HS or SCR group, S. sanguis strain BD11320
was inoculated into the oral cavity of germ-free IQI mice. These groups
were designated as the HS/bac or SCR/bac group, respectively. Bacterial
suspension (103, 105, 107/mouse) was deposited intraorally through a
soft polyethylene catheter. Immediately after inoculation the catheter
was removed and no further manipulations were performed. Control mice
received PBS. After bacterial inoculation, salivary samples were collected
from each mouse (1, 3, 7, and 14 days after infection) and suspended
at a concentration of 10% in BHI medium and placed on MS and BHI agar
plates as previously described.4 In this investigation, colonization
was assessed by determining the level at which a strain persisted in
the saliva. The bacteria detection limit for assessment of colonization
was 102 CFU/mL. Culture of oral tissues was carried out directly on
BHI agar. Feces were also obtained for monitoring the quality of the
mice and colonization of S. sanguis. Bacterial counts were performed
as described previously in this article.
(DTH) Reaction Against the Cell Wall
Fourteen days after infection, the mice were challenged
in both hind foot pads with 20 µL of a solution containing 0.5 mg/mL
of the cell wall (final 10 µg/mouse) or PBS. One foot pad (right) received
the antigen and the other (left) PBS. The thickness of the foot pads
(right-left) was measured before and after challenge. Control mice received
only PBS to both foot pads.
Immune Responses Against
A peptide derived the sequence of the human hsp 336351(QPHDLGKVGEVIVTKDD)
that has been reported to stimulate T lymphocytes of patients in Japan19
was produced by the American Peptide Company (California, USA). Four
other peptides, including Brn-3b of retinal ganglion cells and Bes-1
of 95-kD protein in S. sanguis,10 were also used. Common sequences were
observed between Brn-3b (1125; AFSMPHGGSLHVEPK) and Bes-1 (229243;
QPHDLGKVGEVIVTKDD) and between Brn-3b (177189; HHHHHHHQPHQAL)
and Bes-1 (373385; HGDHHHFIPYDKL), respectively. Peptide was coated
on 96 well plates using a peptide coating kit (Takara, Tokyo, Japan).
Antibody titer was estimated by ELISA.
Cytokine assay (IL-2, IL-6, TNF-a, IFN-g) was done by
ELISA. Briefly, samples from the oral soft tissues (approximately 0.1-0.2g)
were aseptically removed from the mice. They were suspended at a concentration
of 0.1 g/mL in RPMI 1640 medium (GIBCO Laboratories, Grand Island, NY)
containing 1% (W/V) 3-([cholamidopropyl]dimethylammonio)-1-propanesulfonate
(CHPA; Wako Pure Chemical Co., Kyoto, Japan) and homogenized by a tissue
homogenizer (Micro Multi Mixer, Ic-Ieda Co., Tokyo, Japan). Homogenates
were left on ice for 1 hour and clarified by centrifuging at 2000 x
g for 20 minutes. The organ extracts were stored at -80°C until cytokine
assays were undertaken. Cytokines were quantified with ELISA kits (Genzyme,
Cambridge, MA). The dose was determined by a standard curve and expressed
as pg/0.1g of tissue.
Intraocular Injection of Bacterial Components and Synthetic
Intraocular injection was performed with modification
of the method of Callegan et al.15 We changed the needle and syringe
from normal to the gas-chromatography type. Cell wall, LTA, MDP, and
synthetic peptides were prepared for animal administration (0.2 µL:
0.2 µg/eye of mouse) and injected into an experimental eye of ICR mice,
respectively. Care was taken to avoid traumatizing the lens during injection.
In the contralateral eyes, the same volume of sterile Hanks balanced
salt solution was injected in the same manner as for the control. Five
animals (5 eyes) were included in each group of the experimental or
Fourteen days after infection, tissue specimens were collected
for histologic examination. Specimens were fixed in 10% buffered neutral
formalin and processed by standard procedures. Sections of paraffin-embedded
tissue were stained with hematoxylin and eosin.
The results were expressed as mean ± standard deviation
(SD). Differences between experimental and control groups were determined
using the Mann-Whitney test and P < 0.05 was taken as the level of
significance. Spearmans rank correlation was used for antibody
titers against synthetic peptides.
Colonization of S. sanguis
Mice were divided into 10 groups as shown in Table 1.
Mice were inoculated with S. sanguis after heat-shock or mechanical
damage on the oral mucosal surface (designated as HS/bac and SCR/bac,
respectively). The group of mice not undergoing any treatment before
the bacterial infection was designated as the Bac group. The HS, SCR,
and control groups corresponded to treatment (HS or SCR) alone and the
negative control group. Inoculum size was shown as log10 number. In
the HS/bac(7) group (inoculum size: 107/mouse), the bacteria colonized
the oral cavity at 107.767.86/mL saliva within 7days after infection
(Table 1). In the HS/bac(5) group (inoculum size: 105/mouse) and HS/bac(3)
group (inoculum size: 103/mouse), colonization was persistent throughout
the observation. There were significant differences in colonization
between the HS/bac and bac groups until 7days after bacterial inoculation
(P < 0.05).
In the SCR/bac(7) group (inoculum size: 107/mouse), the
bacteria colonized the oral cavity at 105.74-7.22/mL saliva. There were
significant differences in colonization between the SCR/bac and bac
groups until 7days after bacterial inoculation (P < 0.05). The number
of bacteria gradually decreased to that observed in control animals.
The bacterial number in the SCR/bac group was lower than that in the
HS/bac group for the observation period.
In the HS, SCR, and control groups, no bacteria (including
S. sanguis) was detected in the saliva. S. sanguis was detected in the
feces. However, the number of bacteria was similar after inoculation,
ie, 7.5 ± 0.16 (log10, mean ± SD) and 7.4 ± 0.14 (log 10, mean ± SD)
in the HS/bac(7) group and bac(7) groups, respectively. The other HS/bac(5),
HS/bac(3), bac(5), or bac(3) groups had a similar number of bacteria
in their feces.
DTH Response Against S.
The DTH reaction was used to study T cell response to
S. sanguis cell wall antigen in vivo. Ten days after infection, DTH
reaction was induced by injecting S. sanguis antigens into the hind
foot pad (Fig. 1). Foot pad swelling was monitored for 24 hours. DTH
reaction in the HS/bac groups was significantly higher than that of
the others, including control animals (P < 0.05).
Antibody Response Against
IgG antibody titer of the tested sera against the synthetic
peptides was assayed by ELISA (Fig. 2). As shown in Fig. 2A, B, and
C, S. sanguis infection induced an antibody response against the peptides
hsp 336351, Brn-3b 1125, and Bes-1 229243, respectively.
There were significant differences between the HS/bac or bac group and
control animals (P < 0.05). Antibody titer was dependent on the inoculum
size of S. sanguis, especially in the response against hsp 336351
(Fig. 2A). The HS/bac group showed a higher antibody response against
Brn-3b 1125 and Bes-1 229243, but there was no significant
difference among the HS/bac, bac, and SCR/bac groups. In these groups,
antibody responses against Brn-3b 177189 and Bes-1 373385
(different epitopes from Brn-3b and Bes-1) were also seen. No antibody
response was observed in the HS, SCR, and control groups. Correlation
among the antibody responses against hsp, Brn-3b, and Bes-1 was recognized
(P < 0.01). Correlation coefficient in the antibody response ranged
from 0.581 to 0.861, as shown in Table 2.
Detection of Cytokine
Inflammatory cytokines such as IL-2, IL-6, IFN-g, and
TNF-a were detected in oral tissue of the mice infected with S. sanguis
(Table 3). The HS/bac group showed strong local cytokine responses.
However, the SCR/bac and bac groups showed only minimal cytokine responses,
with some levels being similar to those in control animals.
Histopathologic Examination in
Gnotobiotic Mice With S.
Oral tissues of the HS/bac group showed continuous severe
inflammation with cellular infiltration. Microhemorrhages and edematous
changes of the capillary endothelia were observed in the group. A mouse,
which died at 5 days after 107 bacterial inoculation, showed severe
intestinal and genital ulcerations with associated bleeding. Skin lesions
were observed in the HS/bac group. An erythema arose at the site of
inoculation of the bacteria and seemed to be related to severity of
oral ulceration. The skin lesion continued to enlarge until 7 days after
bacterial inoculation, after which it gradually disappeared. The peripheral
ring-like erythema lesions were approximately 1-2 cm in width, with
a range of 0.54 cm, but differed from erythema nodosum or erythema
multiforme seen in the patients with BD. These lesions were characterized
in the HS/bac group.
Oral tissue in the SCR/bac group showed mild inflammation.
Oral epithelial degeneration and some polymorphonuclear leukocytes were
seen in the lesion. The skin lesion was limited around the trauma area.
No systemic diseases were observed in these animals.
The Bac group showed only limited oral epithelial degeneration
with infiltration of a small number of polymorphonuclear leukocytes.
The mice had no systemic diseases. There were no obvious clinical signs
(weight loss at the site of necropsy, diarrhea, and others) as a result
of the stress alone.
Mice inoculated with S. sanguis showed mild anterior segment
inflammation such as ocular lesions (Fig. 3). This lesion was not only
in HS/bac group, but also the bac group and SCR/bac group. However,
we could not induce posterior segment inflammation. A microscopic examination
revealed mild polymorphonuclear leukocyte infiltration in the affected
sites. The lesion was not observed in control animals.
Contribution of Bacterial Components
and Peptides to Intraocular Inflammation
To assess the relative contributions of bacterial components
and peptides to ocular inflammogenicity, cell wall, LTA, MDP, and synthetic
peptides were injected into the eye. CW, LTA, and MDP showed ocular
inflammogenicity at 24 hours (Fig 4). The inflammatory cells and fibrin
were observed in the anterior chamber. Hemorrhages were also seen at
the affected sites. At 6 hours after injection of these bacterial products,
the number of inflammatory cells in the eye was slight. Synthetic peptide
Brn-3b induced cellular inflammation at 24 hours (Fig. 5) but not 6
hours. After injection of hsp336351 or Bes-1, histologic features
were similar to their controls (Fig. 5).
The possibility of a role for S. sanguis in BD has been
raised by several observations.3,5,9,19 In this study, it is clear that
S. sanguis infection after oral heat trauma in germ-free mice can induce
oral and ocular diseases similar to BD. Thus, it seems that the severity
of oral tissue damage is important to trigger the disease. After colonization,
another step such as ulcer formation could be required before the potentially
harmful systemic events can occur. We think at least the following three
conditions must be in place: 1) significant antigenic mimicry between
the microbe and host, 2) an abnormal cellular and humoral response on
the part of the host to the microbial antigens cross-reactive with tissue
antigens, and 3) genetic factors that favor an abnormal host response
to cross-reactive antigens. The trauma appears to be the turning point
as to whether the S. sanguis infection is limited to a local site or
expanded to a systemic level.
In this study, mice colonized by the organisms showed
a delayed hypersensitivity foot pad reaction against the bacterial antigen.
Oral trauma such as heat damage can enhance the entrance of the bacteria;
in the mouse model, the number of colony counts was significantly elevated.
DTH reaction against the cell wall, high inflammatory cytokine levels,
and severe damage of oral tissue were demonstrated only in the HS/bac
group. High levels of colonization would lead to severe inflammatory
Bacterial adherence to mammalian cells is thought to be
the first step in the process leading to infection. We described that
the adhesion of S. sanguis to the buccal epithelial cells from patients
with BD was different from that in healthy control subjects.20 The epithelial
cells exposed to S. sanguis exhibited varying and identifiable degrees
of adhesiveness for the organisms in patients with BD. S. sanguis easily
adhered to the degenerative cells. In our mouse model, HS treatment
enhanced the bacterial colonization. This fact could be reflected in
patients with BD.
animals showed oral ulceration with mild secondary anterior segment
inflammation but no other signs of BD. Only one mouse, which died 5
days after infection in the HS/bac(7) group, showed severe intestinal
and genital ulcerations. Skin lesions in the mice were different from
those in patients with BD. The human major histocompatibility complex
encodes highly polymorphic HLA responsible for antigen presentation
to T cells, and BD is known to be strongly associated with a particular
HLA-B allele, HLA-B51.21 Mice do not possess these disease-susceptible
genes. In the progressive stage such as posterior segment inflammation,
these disease-susceptible genes could be needed.
It has been reported that hsp, specifically amino acid
sequence 336351, is an important antigen.8,9,22,23 Our mouse model
showed infection of S. sanguis induced an immune response against the
synthetic peptide. This response depended on bacterial inoculum size.
Involvement of hsp in autoimmune responses depends on two criteria;
first, hsp needs to be expressed by cells of the target organ in a different
form from that at other tissue sites to allow organ-specific recognition
by T cells and antibodies, and second, control of natural regulatory
mechanisms for organ-specific inflammation must be disturbed.7 Pathogenic
role of hsp peptides has not been accepted.2 In this experiment, direct
ocular inflammogenicity was not observed in mice injected with hsp336351.
However, the specificity of the hsp peptides for BD can be applied as
a diagnostic test.8 In a recent study, we extracted cellular DNA of
S. sanguis from a patient with BD and cloned the fragments.10 At least
two peptides were recognized as antigenic common determinants in both
human cases6,10 and this mouse model. Antibody titer against Brn-3b
and Bes-1 correlated with the titer against the hsp peptide. Our results
showed the molecular mimic peptides can induce autoimmune-like responses.
During retinogenesis, retinal progenitors undergo a series
of changes in competence to give rise to the seven classes of retinal
cells present in the adult retina.24 The ganglion cells are the sole
output neurons in the retina that relay light information detected by
the photoreceptors to the brain. It has been suggested that there are
critical roles for the Brn-3 POU domain transcription in the promotion
of ganglion cell differentiation and in maintenance of differentiated
Brn3b can also mediate some of the effects that FGF2,
TGFß1, and retinoic acid have on neurons.25 Antibody against Brn-3b
could mean that antibody-mediated immunopathogenesis is present in BD
or that there could be only crossreactive results after S. sanguis infection.
We do not know Brn-3b expression or its function in BD. Brn-3b showed
ocular inflammogenicity but not Bes-1. There could be some association
between Brn-3b abnormalities and progression of neuro-BD. Further studies
The flora and metabolites have been found to contribute
to health and diseases. Before this experiment, we examined the potential
ability of colonization various anaerobes in the members of normal human
flora. They colonized in the germ-free mouse but did not induce mucocutaneous
ocular lesions. Mono-infected S. sanguis in this study (without any
flora) induced lesions similar to BD through colonization on oral mucosal
surfaces. This model can help us understand some of the unusual and
as yet unexplained features of BD. One of the key aspects of the model
is the prominent role played by environmental factors in the early stages.
The tissue tropism of the disease could result from restrict exposure
to environmental trigger through some bacterial agents. Cumulative exposure
resulting in toxic levels being achieved only after many years could
explain the age of onset of BD.
The tentative findings provide that a part of S. sanguis
is pathogenic and can be a causative agent for BD. Molecular mimic peptides
can be implicated in the pathogenesis of BD. Cell wall of the bacteria
shows direct ocular inflammogenicity.
The authors thank Dr. Lynn Hyghes, Department of Bacteriology,
Okayama University Graduate School of Medicine and Dentistry, for the
critical reading of the manuscript. Supported by a research grant from
the Behçets Disease Research Committee of Japan of the Ministry
of Health and Welfare of Japan.
Sakane T, Takeno M, Suzuki N, et al: Behçets disease. N
Engl J Med 341:12841291, 1999.
Kaklamani VG, Variopoulos G, Kaklamanis PG: Behçets disease.
Semin Arthritis Rheum 27:197217, 1998.
Isogai E, Ohno S, Takeshi K, et al: Close association of Streptococcus
sanguis uncommon serotypes with Behçets disease. Bifidobacteria
Microflora 9:2741, 1990.
Isogai E, Ohno S, Kotake S, et al: Chemiluminescence of neutrophils
from patients with Behçets disease and its correlation with an
increased proportion of uncommon serotypes of Streptococcus sanguis
in the oral flora. Arch Oral Biol 35:4348, 1990.
Mizushima Y (The Behçets Disease Research Committee of
Japan): Skin hypersensitivity to Streptococcus sanguis and the induction
of symptoms by the antigens in Behçets diseaseA multicenter
study. J Rheumatol 16:506511, 1989.
Isogai E, Isogai H, Kotake S, et al: Antibody cross reactivity
from sera of patients with Behçets disease with synthetic peptides
that have homologies with proteins from Streptococcus sanguis. J Appl
Res 2:17, 2002. (http://www.jrnlappliedresearch.com/index.htm)
Zügel U, Kaufmann HE: Role of heat shock proteins in protection
from and pathogenesis of infectious diseases. Clin Microbiol Rev 12:1939,
Hasan A, Fortune F, Wilson A, et al: Role of g d T cells in pathogenesis
and diagnosis of Behçets disease. Lancet 347:789794, 1996.
Lehner T, Lavery E, Smith R, et al: Association between the 65-kilodalton
heat shock protein, Streptococcus sanguis, and the corresponding antibodies
in Behçets disease. Infect Immunol 59:14341441, 1991.
Yoshikawa K, Kotake S, Kubota T, et al: Cloning and sequencing
of bes-1 gene encoding the immunogenic antigen of Streptococcus sanguis
KTH-1 isolated from the patients with Behçets disease. Zentbl
Bakteriol 287:449460, 1998.
Liu W, Khare SL, Liang X, et al: All Brn3 genes can promote retinal
ganglion cell differentiation in the chick. Development 127:32373247,
Gan L, Xiang M, Zhou L, et al: POU domain factor Brn-3b is required
for the development of a large set of retinal ganglion cells. Proc Natl
Acad Sci USA 93:39203925, 1996.
Fox A, Hammer ME, Lill P, et al: Experimental uveitis elicited
by peptideglycan-polysaccharide complexes, lipopolysaccharide, and muramyl-dipeptide.
Arch Ophthalmol 102;11631067, 1984.
Heumann D, Barras C, Severin A, et al: Gram-positive cell walls
stimulate synthesis of tumor necrosis factor alpha and interleukin-6
by human monocytes. Infect Immunol 62:27152721, 1994.
Callegan MC, Booth MC, Jett BD, et al: Pathogenesis of Gram-positive
bacterial endophthalmitis. Infect Immunol 67:33483356, 1999.
Narikawa S, Suzuki Y, Takahashi M, et al: Streptococcus oralis
previously identified as uncommon `Streptococcus sanguis in Behçets
disease. Archs Oral Biol 40:685690, 1995.
Yokota K, Hayashi S, Araki Y, et al: Characterization of Streptococcus
sanguis isolated from patients with Behçets disease. Microbiol
Immunol 39:729732, 1995.
Schleifer KH, Kandler O: Peptidoglycan types of bacterial cell
walls and their taxonomic implications. Bacteriol Rev 36:407477,
Kaneko S, Suzuki N, Yamashita N, et al: Characterization of T
cells specific for an epitope of human 60-kD heat shock protein (hsp)
in patients with Behçets disease. Clin Exp Immunol 108:204212,
Isogai, E, Isogai H, Fujii N, et al: Adhesive properties of Streptococcus
sanguis isolated from patients with Behçets disease. Microb Ecol
Health Dis 3:321328, 1990.
Yokota K, Hayashi S, Fujii N, et al: Antibody response to oral
streptococci in Behçets disease. Microbiol Immunol 36:815822,
22. Pervin K, Chiderstone A, Shinnick T, et al: T
cell eptitope expression of mycobacterium and homologous human 65-kilodalton
heat shock protein peptides in short term cell lines from patients with
Behçets disease. J Immunol 151:22732282, 1993.
Mizuki N, Inoko H, Ohno S: Molecular genetics (HLA) of Behçets
disease. Yonsei Medical J 38:333349, 1997.
Cepko CL: The roles of intrinsic and extrinsic cues and bHLH
genes in determination of retinal cell fates. Curr Opin Neurobiol 9:3746,
Wyatt S, Ensor L, Begbie J, et al: NT-3 regulates expression
of Brn-3a but not Brn3b in developing mouse trigeminal sensory neurons.
Molecular Brain Res 55:254264, 1998.
Table 1. Colonization
of S. sanguis to Germ-Free
Number (Log10 CFU/mL)* of S.
in the Saliva After Infection
Size 1 Day 3 Days 7 Days
HS/bac 107 7.76 ± 0.30
7.86 ± 0.56 7.84
± 0.12 6.12 ± 0.77
7.22 ± 0.20
7.02 ± 0.21 6.67 ± 0.15 5.74 ± 0.37
Bac 107 5.95 ± 0.45
6.51 ± 0.12 6.36
± 0.67 5.06 ± 0.37
HS/bac 105 7.40 ± 0.43
8.01 ± 0.02 7.82
± 0.41 6.01 ± 0.64
Bac 105 5.25 ± 0.48
6.22 ± 0.20 5.47
± 0.47 5.96 ± 0.22
HS/bac 103 6.86 ± 0.46
8.03 ± 0.12 7.48
± 0.07 5.90 ± 0.46
Bac 103 5.48 ± 0.72
5.81 ± 0.08 6.28
± 0.25 5.59 ± 0.32
HS 0 0 0 0 0
SCR 0 0 0 0 0
Control 0 0 0 0 0
Table 2. Coefficient
Among the Antibody Responses Against the Synthetic Peptides
Hsp Brn-3b Bes-1 Bes-1 Brn-3b
336351 1125 229243
1125 0.380* 1.000
229243 0.479* 0.496* 1.000
373385 0.165 0.325* 0.257 1.000
177189 0.044 0.567* 0.301 0.264
3. Detection of Cytokines
in Oral Soft Tissue of Germ-Free Mice Infected With S. sanguis BD11320
Cytokine Level* (pg/0.1 g)
Inoculum Size IL-2 IL-6 IFN-g TNF-a
107 43.6 ± 19.1 60.5 ± 40.5 62.0 ± 30.4 56.3
SCR/bac 107 <15.0 17.0 ± 6.7 22.7 ± 8.0 <10.0
Bac 107 <15.0 13.5 ± 8.8 17.2 ± 6.8 16.3 ± 10.8
HS/bac 105 45.2 ± 21.8 74.2 ± 43.4 51.0 ± 30.3 65.0
Bac 105 <15.0 7.9 ± 1.2 8.2 ± 4.7 12.0 ± 4.6
HS/bac 103 75.1 ± 49.3 96.0 ± 72.0 117.6 ± 41.2 101.4
Bac 103 <15.0 11.6 ± 8.3 13.1 ± 7.2 21.7 ± 13.5
HS 0 <15.0 16.1 ± 3.6 15.1 ± 8.4 23.3 ± 24.2
SCR 0 <15.0 <5.0 <5.0 <10.0
Control 0 <15.0 <5.0 <5.0 <10.0
Figure 1. DTH
induction after S. sanguis infection. DTH response of the HS/bac group
is significantly higher than that of each bac group and controls (P
<.05). HS/bac(7), HS/bac(5), Hs/bac(3): inoculum size of S. sanguis
(107, 105, and 103 CFU/mouse, respectively). SCR, scraping. Data
indicate mean ± standard deviation.
Figure 2. IgG
antibody responses against synthetic peptides. (A) Antibody response
against hsp 336351. (B) Antibody response against Brn-3b 1125.
(C) Antibody response against Bes-1,229243. HS/bac(7), HS/bac(5),
HS/bac(3): inoculum of S. sanguis (107,
and 103 CFU/mouse, respectively). SCR, scraping. Data indicate mean ±
standard deviation. Experimental groups are significantly higher than
that of controls.
Figure 3. Mild
iridocyclitis after inoculation with S. sanguis (105 CFU/mouse)
and heat treatment. Infiltration of polymorphonuclear leukocytes is
seen in the lesion (x800).
MDP MDP control
Figure 4. Histopathologic
findings after intraocular injection of CW, LTA, and MDP. Injection
of these materials caused influx inflammatory cells (polymorphonuclear
cells) and fibrin accumulation.
hsp control BRN-3b
Figure 5. Histopathologic findings
after intraocular injection of hsp 336351 (HSP), Brn-3b, and Bes-1.
Brn-3b caused influx inflammatory cells (polymorphonuclear cells) and
fibrin accumulation but hsp 336-351did not.