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RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, BANGALORE,
KARNATAKA
ANNEXURE II
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERTATION
1
NAME OF THE CANDIDATE DR. VIJAYALAXMI M. KOKATNUR
P.M.N.M.
AND ADDRESS
DENTAL
COLLEGE
AND
HOSPITAL, BAGALKOT – 587101
KARNATAKA
2
NAME OF THE INSTITUTION
P.M.N.M.
DENTAL
COLLEGE
AND
HOSPITAL, BAGALKOT – 587101
KARNATAKA
3
COURSE
OF
THE
AND SUBJECT
4
STUDY M.D.S. (MASTER OF DENTAL SURGERY)
PERIODONTICS
DATE OF ADMISSION TO 31st MAY 2010
THE COURSE
5
TITLE OF THE TOPIC
IN VIVO EXPRESSION OF TOLL-LIKE RECEPTOR PROTEIN RP105 IN
CHRONIC AND AGGRESSIVE PERIODONTITIS
BRIEF RESUME OF THE INTENDED WORK
6.1 Need for study
Periodontal disease initiation and progression occurs as a consequence of the host immune
inflammatory response to oral pathogens. Many of the microbial surface protein and
lipopolysaccharide molecules are responsible for eliciting a host immune response, resulting in
local tissue inflammation. The inflammatory infiltrate from the gingival tissue can initiate tissue
and alveolar bone destruction through the activation of several pro-inflammatory cytokines,
including interleukin-1β, tumor necrosis factor-α and interleukin-6.1
Host parasite reactions can be divided into innate immunity and adaptive immunity. Innate
immunity is non-specific. It includes inflammatory response and do not involve immunologic
response.6 The adaptive immune system is composed of highly specialized, systemic cells and
processes that eliminate or prevent pathogenic challenges. The
adaptive
immune
response
provides the vertebrate immune system with the ability to recognize and remember specific
pathogens, and to mount stronger attacks each time the pathogen is encountered.
The recognition of pathogens is mediated by a set of germ-line-encoded receptors that are
referred to as Pattern-recognition receptors. These receptors recognize conserved molecular
patterns or PAMPs (pathogen-associated molecular patterns) shared by large groups of
microorganisms. This recognition system is not only responsible for the first-line microbial
clearance but also plays an instructive role in the adaptive immune response through the release
of inflammatory cytokines and expression of co-stimulatory molecules by APCs (antigen
presenting cells). The family of Toll-like receptors (TLRs) has been found to function as PatternRecognition receptors.3
Recently, 10 human Toll-like receptors (TLRs) have been identified, and the TLRs have
been shown to play an essential role in response to bacterial cell surface components including
lipopolysaccharide.4 Toll-like receptor 2 recognizes peptidoglycan, Toll-like receptor 3
recognizes viral double-stranded RNA, Toll-like receptor 4 recognizes lipopolysaccharide, Tolllike receptor 5 recognizes viral single-stranded RNA, and Toll-like receptor 9 recognizes
bacterial DNA.5
RP105 is a B-cell surface molecule that has been recently assigned as CD180.8
They are associated with small
molecules called MD-2 and MD-1,respectively ,to form
heterodimers (TLR4/MD-2 and RP105/MD-1) and function as recognition/signaling molecules
of
lipopolysaccharide(LPS), a member component of Gram-negative bacteria. The B-cell
activation pathway using RP105 is conserved in humans.6 Moreover, the anti-RP105 MoAb
activates B-cell, leading to increase in cell size, expression of co Stimulatory molecule CD80 and
DNA synthesis. Earlier reported studies have shown that RP105, a homolog whose expression
mirrors that of TLR4 on antigen presenting cells, is a negative regulator of TLR4 signaling.6
Although the activation of proinflammatory responses through TLRs is critical for host defense,
excessive inappropriate inflammation can itself be maladaptive. RP105 joins a growing group
of molecules that can inhibit TLR signaling. Regulation of TLR expression provides one point of
control, as does the complex phenomenon of endotoxin tolerance. Several direct, negative
regulators of TLR signaling have been found, including MyD88s, IRAK-M, Etc. Among
these,RP105 stands out for its apparent specificity for the inhibition of TLR4 signaling.7TLR4
may also stand out among the TLRs for its ability to signal in response to a variety of
endogenous `danger signals’.9 The ability of TLR4 to recognize endogenous heat-shock proteins
and extracellular matrix components `unmasked’ by tissue injury suggests the possibility that
RP105 may well be of special importance in down modulation of the injurious inflammatory
responses noted in the systemic inflammatory responses and in autoimmune disease. Hence, the
purpose of the present study is to quantifying the expression of RP105 in gingival tissues of
chronic and aggressive periodontitis patients using Immunohistochemistry technique.
6.2 Review of literature
A study was done to show that human MD-1 is also associated with human RP105 and has
an important role in cell surface expression of RP105 .The monoclonal antibody (MoAB) that
recognizes human RP105 was also described. An immunohistochemical study was carried out
where in the normal tonsil tissues were embedded in Tissue-Tek II OCT compound (Miles Inc,
E1khart. IN), frozen in liquid nitrogen, and stored at -800C.Acetone-fixed cryostat sections of
4µm where incubated with the anti-RP105 MoAB MHR73. After washes, ENVISION+/HRP
(DAKO Japan,, Inc, Tokyo, Japan)was added.RP105 was finally visualized with 3,3’diaminobenzidine.Histological studies showed that RP105 is expressed mainly on mature B cells
in mantel zones. Germinal center cells are either dull or negative.RP105 is thus a novel B-cell
marker that is preferentially expressed on mature B-cells. Moreover, pathway using RP105 is
conserved in humans.6
RP105 was discovered originally in mouse B cells. In this quoted study ,an antibody raised
against the surface of mature B cells was found to drive B cell proliferation and protection
from subsequent radiation - or dexamethasone induced apoptosis. Cloning of human and
mouse RP105 revealed a type 1 transmembrane protein structurally similar to TLRs, with an
extracellular- rich repeat domain and the pattern of juxtamembrane cystein residues
conserved among the TLRs. The analysis of mice homozygous for the null mutation in the
RP105 gene revealed impaired proliferative and humeral immune responses of RP105deficient B cells to LPS. Using
originally LPS-unresponsive
Ba/F3
cells
expressing
exogenous TLR4 and RP105 in LPS – induced nuclear factor kB activation. These data
suggest the existence of the TLR4-RP105 signaling module in the LPS- induced B cell
activation.2
In a study, a negative regulation of Toll-like receptor 4 signaling by the Toll-like
receptor homolog RP105 was shown in Human Embryonic Kidney cells (HEK 293 cells).The
effects of RP105-MD-1 expression on LPS- driven TLR4 signaling was examined. Notably,
RP105 expression inhibited TLR4-driven IL-8 production by HEK 293 cells in a dose–dependent
manner. Furthermore, RP105-mediated inhibition of LPS-driven IL-8 production was associated
with inhibition of LPS-driven transactivation factor NF-kB, suggesting that modulation of IL-8
production by RP105 was `upstream’ of NF-kB activation. Later the specificity of RP105mediated suppression of proinflammatory signaling was examined which showed that RP105 did
not inhibit IL-1 or TLR2-driven IL-8 production, indeed, RP105 over expression led to variable
augmentation of TLR2-mediated IL-8 production in some experiments. Thus, RP105- mediated
suppression shows specificity among the TLR-IL-1 receptor family.7
In a study, the gene expression profile in human periodontal ligament cells
(HPDLCs) in response to TLR4 ligands, LPS treatment was analyzed by microarray analysis. It
was observed that LPS treatment could induce TLR4 activation, increase the gene expression of
colony stimulation factor 2(CSF2), and decrease the gene expression of RP105/LY64 in vitro
.These results suggests the possible involvement of RP105 in TLR4 mediated periodontal
inflammation responses.
A study was conducted to explore the in vivo expression of TLR-2, TLR-4, CSF2 and
LY64 (Ly64 and LY64 are genes related to RP105) in chronic periodontitis patients, who are
systemically healthy and non-smokers. RT-PCR was performed to determine the mRNA
expression levels ofTLR2, TLR4, CSF2 and RP105 in gingival samples. In this study the gene
expression levels of CSF2 increased whereas LY64 mRNA expression decreased. Further studies
should be conducted to ascertain whether inflammation was the only reason for the decrease of
LY64 expression in chronic periodontitis patients.1
A study was carried out to see the altered expression of TLR homolog RP105 on
monocytes hypersensitivity to LPS in patients with primary biliary cirrhosis, wherein invitro
responses to TLR ligand, LPS, using peripheral blood mononuclear cells and monocytes from 25
patients with PBC, 10 patients with chronic viral hepatitis(CVH), and 20 healthy individuals was
investigated. After stimulation with LPS, the results significantly showed higher amounts of IL1β, IL-6, and IL-8 production in PBC patients. Through the TLR signaling pathway, activation
of NF-kB and expression of MyD88 mRNA were significantly increased in PBC patients and the
level of TLR4 expression was significantly increased on PBC monocytes as compared with CVH
patients and controls. Of significance, the surface expression of RP105, which has recently been
shown to be involved in negative regulation of TLR4 signaling, on PBC monocytes was
significantly decreased in comparison with CVH patients and controls.11
6.3 Aims & Objective
1. To quantify the expression of RP105 in gingival tissues of subjects with healthy
periodontium
2. To compare the expression levels of RP105 in gingival tissues of healthy controls and
those with aggressive periodontitis.
3. To correlate the expression levels of RP105 with severity of periodontal diseases.
MATERIALS AND METHODS
7.1 Source of data
Sixty five patients will be selected from the outpatient Department of Periodontics, PMNM
Dental College and Hospital, Bagalkot.
7.2 Methods of collection of data
The study will be carried out on 15 subjects with healthy periodontium, 25 patients with
chronic periodontitis and 25 patients with aggressive periodontitis. Gingival tissue samples from
the selected subjects will be obtained for the analysis of RP105 expression. Samples are grouped
as follows:
Group I : 15 gingival tissue samples from subjects with healthy periodontium
Group II: 25gingival tissue samples from patients with chronic periodontitis
Group III: 25 gingival tissue samples from patients with aggressive periodontitis
Gingival tissue specimens for group I will be obtained during tooth extraction due to
orthodontic treatment purpose and during third molar extraction. For groups II and III, specimens
will be obtained during periodontal surgery. Specimens obtained will be placed in buffer solution
and will be sent to laboratory for quantification of RP105 by using immunohistochemistry
method.
INCLUSION CRITERIA
1) Group I: Healthy subjects showing absence of clinical manifestations of periodontal disease,
at least 15 teeth and present no history of periodontal treatment for the past six months.
2) Group II: Chronic periodontitis patients with periodontal pockets >5mm.
3) Group III: Aggressive periodontitis patients.
All the patients should be systemically healthy and none should have received
periodontal treatment for at least 6 months prior to sampling and recording.
EXCLUSION CRITERIA

Smokers

Systemic diseases like diabetes mellitus, hepatitis, and HIV infection known
to
influence the periodontal disease.

Diseases of oral hard and soft tissue except caries and periodontitis.

Chronic usage of antibiotics and analgesics within three months prior to study.

Pregnant and lactating female.
All potential participants were explained the need and design of the study. Only those subjects
who gave consent for the study were included after obtaining an informed consent.
CLINICAL EXAMINATION TO ASSESS THE PERIODONTAL CONDITION

Gingival index(Loe & sillness)

Probing pocket depths(using William’s graduated periodontal probe)

Clinical attachment loss
LABORATORY INVESTIGATIONS
Gingival tissue specimens will be sent to the laboratory for quantification of RP105 using
immunohistochemistry. Monoclonal anti-human RP105 antibody kit will be procured from
Biolegend, USA
STATISTICAL ANALYSIS
The results will be statistically analyzed using the following methods.

One way ANOVA- It is used to compare values among three groups.

Mann-Whitney test or t-test - It is used to compare values among two groups.
7.3 DOES THE STUDY REQUIRE ANY INVESTIGATIONS OR
INTERVENTIONS
TO BE CONDUCTED IN THE PATIENTS?
Yes. Gingival tissue samples have to be obtained from the patients.
7.4 HAS ETHICAL CLEARANCE BEEN OBTAINED FROM THE INSTITUTION?
Yes. The copy of ethical clearance certificate is attached.
REFERENCES
1. D-L Liu, Y Sun, M-Z Zhang, Q-M Guo, R Shu. In vivo expression of Toll-like receptor
2, Toll-like receptor 4, CSF2 and LY64 IN Chinese chronic periodontitis patients. Oral
Diseases, 2010 (16): 343-50.
2. Hirotaka Ogata, I- hsin Su, Kensuke Miyake, Yoshinori Nagai, Sachiko Akashi,
Alexander Taraskhovsky, et al. The Toll-like receptor protein RP105 regulates
lipopolysaccharide signaling in B cells. J Exp Med, 2000 Jul 3; 192(1)23-30.
3. Kirkwood KL, Cirelli J A, Rogers JE, Giannobile WV. Novel host response
therapeutic approaches to treat periodontal diseases. Periodontol 2000 2007; (43) 294315.
4. Medzhitov R, Preston- Hurlburt P, Janeway CA Jr. A human homologue of the
Drosophila Toll protein signals activation of adaptive immunity. Nature 1997 ;( 388)
394-7.
5. Mahanonda R, Pichayangkul S. Toll-like receptors and their role in periodontal health
and disease. Periodontol 2000 2007; (43) 41-55.
6. Miura Y, Shimazu R, Miyake K, Sachiko Akashi, Hirakata Ogata, Masao Kimoto. RP105
is associated with MD-1 and transmits an activation signal in human B cells. Blood 1998
;( 92) 2815-22.
7. Senad Divanovic, Rajat Madan, Christopher L Karp, Aurelian Trompette, Sowsan F
Atabani, Robert W Finberg, et al. Negative regulation of Toll-like receptor 4 signaling by
the Toll-like receptor homologue RP105. Nature Immunol 2005 April 26; 6(6):571-4.
8. Nagai Y, Shimazu R, Ogata H, Akashi S, Sudo K, et al. Requirement for MD-1 in cell
surface expression of RP105/CD180 and B-cell responsiveness to lipopolysaccharide.
Blood 2002; 99(5):1699-170.
9. Takeda K, Kaisho T & Akira S. Toll-like receptors. Annu. Rev Immunol, (21)335-376.
10. Nagai Y, Kobayashi T, Motoi Y, Ishiguro, et al. The radiprotective 105/MD-1 complex
links TLR2 and TLR4/MD-2 in antibody response to microbial membranes. J Immunol.
(174):7043-49.
11.Yutaka Honda, Satoshi Yamagiwa, Yasunobu Matsuda, Masaaki Takamura, Tamaki
Ichida, Yutaka Aoyagi. Altered expression of TLR homolog RP105 on monocytes
hypersensitive to LPS in patients with primary bilary cirrhosis. J Hepatol, 2010 June 10;
47(3):404-11