1. Preliminary evidence for a link between periodontal disease and Alzheimer’s disease (AD)
Sophie Poole, Oral & Dental Sciences Research Group, School of Postgraduate Medical & Dental Education,
UCLan
Good morning, I’m Sophie Poole from the University of Central Lancashire and today I’d like to talk to you about the results from my on going PhD project which has presented with preliminary evidence for a link between periodontal and Alzheimer’s disease.

2. Introduction
Alzheimer’s disease & Periodontal disease(s) are complex, multifactorial, progressive & inflammatory.
Clinical and epidemiological studies suggest that periodontitis may be a risk factor for Alzheimer’s disease.
Study purpose - to find evidence for a link between periodontal disease and AD.
There are an estimated 35.6 million people living with dementia in the world (World Alzheimer’s Report)
Rationale: Periodontal therapy from early age may delay onset and reduce the figure of AD patients significantly
I’m sure that we are all aware that AD is a complex, multifactorial progressive and above all inflammatory disease. However, not everyone will be aware that AD shares these four properties with PD.
Studies have shown the potential of PD as a risk factor for AD and therefore, I am looking to find further evidence for a link between the two conditions.
There are an estimated 35.6 million people diagnosed with dementia in the uk with the main contributor being sporadic AD yet the cause of sporadic AD remains under investigation, whereas the cause for PD is known.
Therefore, if a significant link is established dental intervention may have a huge impact on the disease prevalence dramatically decreasing an individuals risk of developing AD or potentially reducing the progression of the disease.
So what is PD?

3. Chronic Periodontal disease
control
Clinical features
Gingival
recession
Gingivitis
Plaque & Calculus
Pus
The main cause
The lesion
Porphyromonas gingivalis (Pg),
Tannerella forsythia (Tf), and
Treponema denticola (Td) are
intimately associated with the disease.
200 nm
0.25 mm
Pg, Tf, Td
X-ray shows a periodontal
Pocket (wound) – typically 109
bacteria
Unlike AD PD can affect individual from a very young age. As you can see there is a visible difference between a healthy mouth and that of an individual with chronic PD. You can see all the puss and bacteria which form deep into these pockets in this highly vascular tissue thereby making it easy to understand how bacteria can enter the blood stream and contribute towards systemic inflammation. These transient bacteriemias can occur through everyday activities such as chewing food or brushing teeth.
There are 3 main bacteria associated with PD known as the red complex bacteria; these being Porphyromonas gingivalis, Treponema denticola and Tanerella forsythia. However in this presentation I’m focusing on P. gingivalis
These species of bacteria live in these pockets and destroy the host tissue. This is the lesion of PD (pocket)
Bacteraemia: ref’s: Forner et al., 2006 J Clin Periodontol 33:

4. Alzheimer’s disease Specific-lesion Inflammation
Pathological features
Specific-lesion
Inflammation
Astrocytes & microglia
The lesion of AD is very different from that of PD as I’m sure we’re all familiar with the pathological hallmarks such as; the amyloid plaques, neurofibrillary tangles and most important for my current focus inflammation involving glial cells.
At this point I wish to acknowledge the “Brains for Dementia Research” tissue bank for kindly supplying me with specimens from both sporadic AD cases and non-AD age matched controls.
Brains for Dementia Research: supplied peri-ventricular cerebral tissue,
N = 10 sporadic AD cases and 5 = non-AD age matched controls

5. 4/10 AD cases show cellular immunolabelling to Pg-endotoxin(s)
Cell surface membrane labelling with anti-Pg (LPS)
CD14….x63
All specimens were received snap frozen. Cryosectioned and then labelled using an antibody specific to the PD pathogen P. gingivalis. After screening all cases I identified 4 /10 of the AD cases presented with positive cellular staining as shown by the green labelling surrounding the red nuclei. Whereas the other 6 AD cases and the 5 non-AD controls remained negative.
The labelling was restricted to the cell surface, as can be seen when compared with a cell surface marker such as CD14.
CD14 is expressed on most of the CNS cells (neurons, astrocytes and microglia) and functions as a phagocytic receptor for pathogen associated molecular patterns e.g. LPS. LPS = bacterial cell wall lipopolysaccharide.
We believe these are glial cells and in order to confirm if glial cells can respond to P. gingivalis we turned to tissue culture
Red = PI nuclear stain. Green = mo α pg1B5 detected by FITC conjugated secondary detection system
Note: the cellular staining was only observed in 4 /10 AD cases only and NOT in the controls brains. However, there are only a few cells that are positive/case in 4/10 cases.

6. In-vitro assimilation
SVGp12 in culture
Negative control
Anti-GFAP labelling
CD14 labelling
We choose the human glioblastoma cell line , SVGp12.
From this slide you can see the general characteristics of these cells, they grow well in culture and having proven that there is no non-specific labelling we can see that the cells present with GFAP and CD14 labelling as expected.

7. Glial cells can adsorb LPS
Duel labelling
Red = GFAP, Green = LPS and nuclei = blue 24 52 31 17 12 76 38
Control med only
Spent med strain
E. Coli LPS only
SGVp12 untreated cells
SGVp12 treated with
33277 spent med
anti-Pg (1B5) labelling
on cells challenged with
control Pg medium
anti-Pg (1B5) labelling
on cells challenged with
Pg spent medium
These cells were then challenged with spent medium from P. gingivalis strain for 48 hours
spent medium is fluid in which the bacteria has been cultured and then removed leaving endotoxins intact
These cell were analysed using the same anti-P. gingivalis antibody as used on the human brain specimens.
The results present with surface membrane labelling as can be seen by the green labelling on this image, which is further supported by the immunoblot whereby the control group of cells remained negative and the test group appeared to adsorb the LPS as can be seen by this characteristic banding pattern.
Having got these results we were satisfied that glial cells can adsorb LPS then revisited the human cases using these lysates as positive and negative controls

8. LPS in AD cases 24 52 38 31 17 12 76
Control med only
Spent med strain
E. Coli LPS only
SGVp12 untreated cells
SGVp12 spent med 33277
AD brain from case 3
AD brain from case 5
AD brain from case 8
AD brain from case 10
This blot shows the controls generated from our in vitro study alongside the four human AD cases which were positive when screened for P. gingivalis.
It’s clear from this blot that the cases have shown a positive result corresponding to the LPS adsorbed by the cells thereby fully supporting our immunofluorescent data.
Loading control

9. NO LPS in NON-AD controls24 52 38 31 17 12 76
Loading control
Control med only
Spent med strain
SGVp12 untreated cells
SGVp12 spent med 33277
Non-AD cont. brain 1
Non-AD cont. brain 2
Non-AD cont. brain 3
Non-AD cont. brain 4
Non-AD cont. brain 5
Alongisde this a complementary blot using lysates prepared from the non-AD control brain was also tested. As you can see the controls generated from in-vitro, remained consistent, whereas the tissue lysates from the non-AD control brains remained negative. This result further supports our IF findings that 4/10 AD cases are positive for P. gingivalis LPS whereas all 5 of the non-AD controls remained negative.
These results are very encouraging although it is still important to determine how these pathogens or their virulence factors are entering the brain.

10. Entry to the brain: Vascular channels
The particles positively labeled with anti-Pg antibody are indicated in green (FITC) from an AD brain. The green staining was observed in the brain tissue and within blood vessels as shown by phase image overlaid on the dark field image.
One clue come in the form of these granular extracellular aggregates in the brain which also labelled positive with the same antibody. They were found in a number of vascular channels including some intra venous sinuses as is demonstrated by this phase image overlay on the dark field image. Although these were not present in all 4/10 cases.

11. Conclusions
Immunolabelling: 4/10 AD cases show P. gingivalis LPS in the brain.
The LPS from P. gingivalis strains does enter the brains of dementia patients and the likely route is the vascular channels.
In vitro study using the SV40 immortalised glial cell line (SVGp12) confirms that glial cells can adsorb LPS.
AD patients appear to be susceptible to PD pathogens and/ or their virulence factors
In conclusion the immunolabelling using two different methods has shown that 4/10 AD cases show P. gingivalis LPS in the brain. A possible route appears to be connected with larger (veins) vessels.
Our findings are further supported by in vitro work using a glial cell line demonstrating that human glial cells can adsorb P. gingivalis LPS
Therefore, our work to date suggests that some AD patients appear to be more susceptible to PD pathogens and/ or their virulence factors.

12. Acknowledgements
UCLan: Dean, Prof. StJohn Crean (PI & DoS), Dr. Sim Singhrao, Associate prof. L. Kesavalu (supervisors)
Prof. M. Curtis, for the essential reagents:
(Control and spent media from Pg strains and W50)
(Anti-P. gingivalis antibody clone 1B5 and anti-gingipains antibody clone 1A1)
Images:
Pg image c/o Prof. M. Curtis (UK), Tf image c/o Dr Stafford (UK) and
Td image c/o Prof. J. D. Radolf (USA)
Thank you.