A hyperactive neutrophil phenotype in ptients with refractory periodontitis

1. Longitudinal effect of non-surgical
treatment and systemic metronidazole for
1 week in smokers and non-smokers with
refractory periodontitis; A 5-Year study
Birgitta Soder et al., 1999

2. Introduction
The rate and severity of tissue destruction reflect the virulence of the bacteria and host
response.
Soder et al. showed that smoking was Sig. correlated to the number of deep pockets.
Smoking is one of the risk factors for periodontal disease.
Periodontitis is the mixture of disease, caused by the present of microorganism.

3. Introduction
Advance periodontitis does not always response to conventional
Give systemically or locallyATB to improve the success rate of periodontal therapy
• In short-term studies, it has been shown that metronidazole, when systemically
administered after debridement, resulted in treatment benefits including less need for
surgical intervention.

4. Introduction
Metronidazole is specific to anaerobes, bactericidal in low concentrations for anaerobe (such
as bacteroides, fusobacteria and treponemes.
Metronidazole can prenetrates into GCF (attains levels similar to in serum and saliva) and this
concentrations which in vitro can inhibit periodontal microorganisms.

5. Material and methods
Patient selection
• 100 non-responders  for drop-out study.
• 144 subjects need periodontal treatment agreed to participate
• 98 of 144 subjects had persisting pathological pockets ≥5 mm
Inclusion and exclusion criteria
• Inclusion: Pt have at least 3 teeth with inflamed pockets, PD ≥ 5 mm, marginal bone loss on
radiographs after initial SRP
• Exclusion: intolerance to metronidazole, neurological disorders, blood dyscrasias, alc. or drug
abuse, pregnancy, lactation.

6. Material and methods
Examinations
• GCF 1 site/ patient for detection of PMNs
• Diluted inTurk’s solution.
• 10 µl of Hank’s solution was injected into the test site  aspirate 10 times by resting the point of a
needle of 25 µl Hamilton microsyringe at orifice of the site.
• PMNs were counted in a Burker chamber (using a microscope)
• Microbial samples were collected by inserting a sterile paperpoint for 30 sec following pockets:
16B, 11B, 25B, 36L, 32B, 46L  then pooled, transfer to transport medium, microbiology laboratory
• Slots’ method: for anerobes & A.a
• Long-wave UV light: +ve P.g, -ve P.i

7. Material and methods
Examinations (cont.)
• Subgingival plaque  curette from the deepest part of the same test sites
• Dark-field microscopic examination  count microorganism (spirochetes, motile
organism, non-motile filament, rods or cocci)
Oral examination
• Charting, PI, GI, CI-S, BOP  by probe with tip diameter of 1 mm, pressure 25 g.
6 sites on each tooth: MB, mid-buccal, DB, ML, mid-lingual, DL
X-ray  FM ( 14 periapical films)

8. Material and methods
Randomization
• Computer-generated randomization list, in block of 10.
Treatment
• SRP
• Metronidazole (400 mg) or placebo. Sig.1x3 for 7 days
• Maintenance 5-year period (every 6 months)
• Surgical intervention (ModifiedWidman flap operation) was indicated when PD ≥ 6mm,
inflamed and PD increased ≥ 2mm btw 2 visits

9. Material and methods
The results: 4 groups
1. Intervention group/ non-smoker
2. Intervention group/ smokers
3. Placebo group / non- smokers
4. Placebo group / smokers
3 types of therapy
1. Non-surgical treatment
2. Non-surgical treatment + metronidazole
3. Non- surgical treatment + metronidazole, and periodontal surgery

10. Results
Drop-Outs
• First 6 mouths  6 pts.
• 1-5 years  28 pts
• After 5 years = 64 pt.  32 intervention  16 non-smokers
 16 smoker
 32 control

11. Results
Smokers and non-smokers in the intervention and placebo groups

12. Results

13. Results
Smokers and non-smokers in the intervention and placebo groups with non-surgical treatment
(Group 1-4 + treatment 1)

14. Results
• Smokers and non-smokers in the intervention and placebo groups treated with periodontal
surgery

15. Results
• Smokers and non-smokers in the intervention and placebo groups treated with periodontal
surgery

16. Results
• Laboratory tests
All decreased

17. Results
• Laboratory tests

18. Discussion
• Smoker responded < Non-smoker
• Sig. diff in the severity of dz. Btw smokerVs Non- smoker
• Adjunctive Metro. on non-surgical tx of smokers in the intervention group was very small
• Metro. did not completely eliminate A.a in this population
• P.g plays an important role in the progression of periodontal dz.
• P.i was very difficult of tx. with and withoutATB
• Spirochetes could not be totally eliminated, but sig. in both groups
• T.d can serve as a marker for recurrence of the dz.
• Smoking  impaired healing of periodontal dz.

19. Discussion
• Neutrophil granulocytes are associated with tissue destruction in a number of chronic
periodontal dz.
• Metronidazole resistance is relatively rare and seems to be due to a decrease in the ability
of the organism to reduce the drug to its active form

20. Conclusion
• Decisive factors in the sustained long-term improvement of patients who
respond satisfactorily to treatment are probably initial scaling and root
planing; a brief course of metronidazole; and regular follow-up
examinations at 6-month intervals for oral hygiene and scaling and root
planing.

21. A Hyperactive Neutrophil
Phenotype in Patients with
Refractory Periodontitis

22. Introduction
• Conventional treatment for most cases of periodontitis is focused on the
removal of bacterial plaque and calculus.
• Periodontitis is a destructive inflammatory disease that adversely affects
the periodontium, or tooth supporting tissues.
Some periodontitis patients do not respond to conventional periodontal
therapy
Refractory periodontitis

23. Introduction
• Neutrophils or polymorphonuclear leukocytes (PMNs) play a vital role in
maintaining periodontal health.
• Host immune response plays a significant role in the development and
progression of RP.
• The author suggest that cause of the destructive process evident in chronic
periodontitis is the “hyperactivity” of neutrophil  overproduction of
antimicrobial and potentially tissue-damaging oxygen free radicals.

24. Introduction
• Aim of this study
• 1. Compare the generation of oxygen radicals in peripheral PMNs from
patients with RAP, CP, and periodontally HCs after stimulation with
phorbol myristate acetate (PMA).
• 2.To examine the phagocytotic ability of the neutrophils.

25. Material and methods
• Patients who presented regularly to
the Faculty of Dentistry, University of
Toronto, for treatment and / or
consultation between September 2003
and September 2006
• Refractory to treatment
• Had been treated by periodontist for
≥ 1 year before refer to the Severe and
Refractory Disease and demonstrated
progressive attachment loss while
patients had adequate maintenance
therapy.

26. Material and methods
• Peripheral blood PMNs were loaded with dihydrorhodamine 123
• stimulated with phorbol 12-myristate 13-acetate (PMA) to measure the
receptor-independent respiratory burst of these key immune cells.
• Phagocytosis via the complement and Fc gamma receptors was also assessed.

27. Results
(NADPH Oxidase Activity as Measured by DHR)
Oxygen radical production after direct
stimulation of intracellular protein kinase C
(PKC) with PMA
“RAP > HC > CP”
Oxygen radical production of RAP and CP
groups compared to HC (tested the same day)
“RAP > CP”

28. Results
(Neutrophil phagocytosis)
Sig. Neutrophil phagocytosis of sRBCs
after complement receptor activation in
RAP subjects compare to CP and HC
groups

29. Discussion
http://www.clinsci.org/content/111/1/1

30. Discussion
• Neutrophil can be activated and generation of ROS
• Receptor ligand-mediated response.
• PMA
• Phosphatidylinositide pathway initiated by phospholipase C, D activation of
PKC by
• Diacylglycerol
• Formation of myoinositol-1,4,5- triphosphate
PKC then phosphorylates several downstream signaling proteins, resulting in events such as
chemotaxis, phagocytosis, respiratory burst, and lysosomal enzyme release.

31. Discussion
• Previous study
• CP patients oxygen radical production when neutrophil were activated via Fc Ɣ receptor
pathway (intrinsic aberrant signaling pathway)
• In this study was supported by our observations in that there was no significant
increase in oxygen radical production evident in the CP group after stimulation
with PMA. Although we did detect a slight decrease in PMA-induced oxygen
radical production in the CP group compared to HCs

32. Question: peripheral ROS and oral ROS (oPMN)?

33. Refractory periodontitis population characterized
by a Hyperactive oral neutrophil phenotype
Guy M. Aboodi et al, 2010

34. Introduction
• Hypothesis  oral neutrophil hyperactivity is related to periodontal disease
severity.
• Used a flow cytometric approach to isolate and analyze oral neutrophil ROS
(oROS) production of a refractory periodontal disease patient population.

35. Introduction
• Oral neutrophil (oPMN) ROS activity during periodontal disease has not been
investigated as thoroughly as peripheral blood neutrophils.
• levels of ROS in GCF of periodontal patients
• levels of 8-hydroxydeoxyguanosine (8-OHdG) of saliva samples.
• 8-OHdG levels  ROS activity  marker for oxidative DNA

36. Introduction
• isolation of oPMNs from oral rinse samples
• rinsing the oral cavity with isotonic solution, followed by filtering out epithelial cells from
the rinse sample using a nylon mesh.
• oral neutrophils have higher ROS levels at baseline compared to peripheral blood
neutrophils, an dthey are able to respond to various types of stimuli.
• The goals of this study
1. to identify the presence of oPMN hyperactivity among RP patients
2. to determine if oPMN hyperactivity is related to a history of periodontal disease
severity in RP patients.

37. Material and method
• Study population
• 13 patients Dx. with RP
• received regular subgingival scaling and prophylaxis every 3 months
• had no contributing systemic conditions, nonsmokers (other than one light smoker; <10
cigarettes per day)
• Study design
• Periodontal examination: PD, BOP,VPI, mobility, furcation involvement, recession
• oROS evaluation  Oral rinse with 5 mL of 0.9% NaCl for 30 sec + expectorate the rinse sample into a 50
mL falcon tube
• ROS evaluation Venous blood 3mL
oROS and ROS were evaluated by flow cytometry testing

38. Material and method
Peripheral blood neutrophil isolation
• Neutrophil isolated by centrifuged at 527 relative centrifugal force for 30 mins
• Harvest from lower of 2 bands
• Wash by centrifugation with phosphate-buffered saline (PBS) at 1,082 relative centrifugal
force for 5 mins at 21 ๐C
• Cells were resuspended in 1 mL of PBS
Oral cellular component isolation
• Filtered through a sterile 40-mm nylon mesh
• Filtered samples were centrifuged at 1,083 relative centrifugal force for 5 minutes at 21oC
• Used of a fluorescent CD-11b antibody, a leukocyte-specific membrane marker for verified the
presence and viability of PMNs (from PMNs, oPMNs)

39. Material and method
Measurements of cellular ROS
• Stained with dihydrorhodamine 123 (DHR)
DHR
rhodamine 123
H2O2
superoxide

40. Results
ROS and oROS levels after stimulate
with PMA

41. Results
Low and high responders
LRs
stimulated oROS% < 45%,
activation potential < 13%.
HRs
stimulated oROS% > 45%,
activation potential > 19%

42. Results
ClinicalAL in the HR group
Mean percentage of sites with AL >5 mm was
found in HR
No Sig. BOP and PD

43. Discussion
• In periodontally healthy patients; compared PMN and oPMN
• Sig. Phagocytic activity
• Similar in bacterial killing abilities
• Diff. levels of activity among salivary, peripheral blood, and GCF neutrophils.
• It was previously shown that oPMN functionality is impaired compared to peripheral blood
PMN maybe because of the functional potential of PMN is maximal in its naive state, before
any stimulation.

44. Discussion
• BOP and PD, which may be considered markers of
active and past disease and influence neutrophil
migration into the crevice, were not significantly
different between 2 groups

45. The gene expression profile in
refractory periodontitis
patients
David M. Kim et al., 2006

46. The gene expression profile in
refractory periodontitis
patients
David M. Kim et al., 2006

47. Introduction
• Refractory periodontitis cannot identified by specific bacteria or other diagnostic
tests
• Hypothesis: patients with refractory periodontitis have multiple upregulated and/or
downregulated genes that might be important in influencing clinical risk.
• “Downhill” patients = loss of 4-9 teeth after periodontal therapy
• “Extremely downhill” = loss of 12-23 teeth after periodontal therapy  small population but
accounted for the most tooth loss.
• Both of downhill and extremely downhill have been referred to refractory periodontitis.

48. Introduction
• The purpose of this study was to use microarray technology to qualitatively and quantitatively
measure gene expression levels
• refractory periodontitis patients and
• periodontally well-maintained patients.

49. Material and methods
Patient selection
• 7 RP
• 7 periodontally well-maintained patients with a past hx. of RP
Collection of CNT samples
• Refractory periodontitis  harvested from the active progressing site of disease
• Periodontally well-maintained patients  from a treated site that required CLP or root coverage
procedure

50. Material and methods
RNA Stabilization and Isolation ofTotal RNA FromTissue Samples
• Submerged in 10 volumes (10 ml reagent per 1mg tissue) of RNA stabilizing reagent.
• RNA isolation kits  isolate total RNA
• Determines quality control and quantitation of total RNA samples by microarray experiments
ReverseTranscription, InVitro cRNA Synthesis, and Microarray Hybridization
• For measure the expression of genes.
Data analysis
• Microarray analysis and real-time PCR analysis

51. Results
• 5 female and 9 male subjects (aged from 48 to 72 years) with ≥ 10 teeth present participated in
this study.
• All subjects were either never-smokers or had stopped smoking at least 1 year before active therapy.
• Were not currently undergoing antibiotic therapy or exhibiting systemic conditions that might affect the
final outcome of this study.
• 68 upregulated and 6 downregulated genes.
Microarray Analysis
• The complete database is comprised of 14 expression measurements of 22,283 genes;
• 7 in the refractory periodontitis condition
• 7 in the periodontally well maintained condition.

52. Results

53. Results
Real-Time PCR analysis
• 5 upregulated genes (lactotransferrin [LTF],
matrix metalloproteinase1 [MMP-1], MMP-3,
interferon induced-15 [IFI-15], and Homo
sapiens hypothetical protein MGC5566)
• 2 downregulated genes (keratin 2A [KRT2A]
and desmocollin-1 [DSC-1])
were randomly selected for further analysis by
real-time PCR
• The analysis by analyze real-time PCR were
compared to microarray data as shown onTable

54. Discussion
• The host response to periodontal disease involves complex interactions between cells,
extracellular matrix, and circulating cytokines.
• Epidemiological studies show that there are significant interactions between genetic
factors and other environmental and demographic factors
• A hyperactive immune response may lead to a hyper production of cytokines and other inflammatory
mediators, which in turn may result in a slow and steady loss of periodontal attachment.
• The results we obtained by both microarray and real-time PCR methods agreed and clearly indicated
that the gene expression profiles in refractory periodontitis patients were different from those of
periodontally well-maintained patients.

55. Discussion
Upregulate gene
MMP-1 and -3
• MMP-1 is the most widely expressed MMP possessing proteolytic activities against fibrillar
collagens (collagen types I, II, and III)
• MMP-3 is a proteoglycanase that is closely related tocollagenase(MMP-1) with a wide range of
substrate specificity.
• Together with other metalloproteases, it can synergistically degrade the major component of
the extracellular matrix.

56. Discussion
Upregulate gene
Interleukin-24 (IL-24) or Melanoma Differentiation Associated Gene-7 (MDA-7)
• IL-24 and MDA-7 belongs to the IL-10 cytokine family (IL-19, -20, -22, -24, and -26).
• IL-10 genetic polymorphisms in the IL-10 gene was recently found to be associated with chronic
periodontitis.
LTF (Lactoferrin)
• Is a natural iron-binding glycoprotein with multifunctional immune regulatory properties

57. Discussion
Upregulate gene
Caspase-10 (CASP-10), Apoptosis-Related Cysteine Protease
• Apoptosis, or programmed cell death, plays a critical role in the regulation of inflammation and host
immune response.

58. Discussion
Downregulate gene
DSC-1
• Maintaining the strength and integrity of epithelial tissues
KRT2A
• Major gene product of keratinocytes and form the intermediate filament cytoskeletal network in
these cells.
Downregulation of DSC-1 and KRT2A genes might make an individual vulnerable to tissue destruction,
such as periodontitis.
The importance of both cell adhesion junctions and the keratins is demonstrated by the large number of
genetic and autoimmune diseases (i.e., pemphigus, pemphigoid, and epidermolysis bullosa) that result
from altered expression or function of these structures.

59. Conclusions
A number of features of gene expression patterns that could be related to upregulation and
downregulation of genes known to be involved in the inflammatory stages of wound healing
and in tissue breakdown.
Several of these genes may be considered useful indicators or diagnostic markers for
periodontal disease susceptibility.