1. Chemotaxis of periodontal neutrophils: do they display aberrant behaviour?
Anice Almuflahi (Aston University, Biological Sciences) Biochemical Society summer vacation
studentship with Dr Melissa Grant (University of Birmingham, School of Dentistry).
The aims of this project were to assess the chemotaxis of neutrophils from both healthy controls
and periodontitis patients to standard cytokine chemoattractants; and neutrophils from healthy
controls to periodontitis patient and control plasma.
Description of the work carried out:
Primaryneutrophils were extractedfromthe blood of healthy volunteers and separated by density
gradientcentrifugation usingPercoll.Theywerecountedandassessed for cell viability using trypan
blue by means of a haemocytometer under a microscope. The neutrophils were then diluted as
appropriate to gain 2.0 x 106
cells/ml. Neutrophils were then applied to a cover slip and left for
20mins to attach. Then the cover slip was placed on an Insall chamber where the different
chemoattractants can be pipetted into the chamber. The 4 chemoattractrants used were RMPI
(negative control), periodontal patient plasma (baseline), treated periodontal patient plasma (1
weekaftertreatment) andfMLP(positivecontrol).The periodontal patient plasma samples needed
to be diluted by a 1:10 dilution in PBS before pipetting them into the Insall chambers. The
movementof the neutrophilswasthen recorded at the viewing bridge of the Insall chamber, using
the invertedZeisschemotaxis microscope andImage Jsoftware over12sintervalsfor7mins.Intotal,
after familiarisation with the assay, neutrophils were isolated from 8 volunteers and 8 pairs of
baseline and treated periodonitis patient plasmas were analysed. From each experiment 10
neutrophilswere chosenatrandomto follow theirpath,giving a total of 80 per experiment and 240
for the whole study.
Afterthe imageswere collected from the Zeiss microscope and processed by the ImageJ software,
the images were subject to statistical analysis using an image processing package called Fiji. To
analyse the datageneratedthe speedof the neutrophilswascalculated(distance from beginning to
end of cell track (A to Z) directly, divided by the time taken); the curvilinear velocity of the
neutrophils was calculated (distance from beginning to end of cell track (A to B to C....to Z) via all
intermediarypoints,dividedbythe time taken);andthe linearity(the ratioof the curvilinearvelocity
over the speed, giving an idea of the straightness of the path travelled).
Results and the outcomes of the studentship:
In this study the speed, curvilinear velocity and linearity of neutrophils under the influence of
negative and positive controls chemoattractants and potential chemoattractants in periodontitis
patient plasma were assessed (figure 1). The standard chemoattractant fMLP (positive control),
significantlyincreasedboththe speedandcurvilinearvelocityof healthyvolunteer neutrophils. This
demonstrated that neutrophils would move along a known chemoattractant gradient. In addition
plasmaobtainedfromperiodontitispatientsbefore treatment also significantly increased both the
speed and curvilinear velocity of health volunteer neutrophils. However once these patients had
beentreatedthiseffectdecreased to near control (RPMI) levels. However, none of the treatments
significantly affected the linearity of the neutrophil cell path. This may be due to the neutrophils
beingfromhealthyvolunteers.The linearityof periodontitis patient neutrophils may be a different
story.
Figure 1.
Bar graphs summarising the speed, curvilinear velocity and linearity of all the cells analysed (data
shows mean + SEM). A Friedman statistical analysis was used with a Dunn’s post test (* p<0.05,
**p<0.01, ***P<0.001) using Graph Pad Prism software.

2. Future directions in which the project can be taken:
It isexcitingtosee thatevenwithsucha small numberof patientsamples(n=8) that significant data
couldbe generated.Totake thisprojectforwardto publicationthe numberof patientsamplescould
be expanded,perhapsto n=20, as the curvilinearvelocity is approaching statistical significance too.
There is something within the baseline plasma that is causing a greater chemoattraction than the
periodontitispatientsthathave beentreated. Future studies could try and address what this might
be, it is likely to be associated with the inflammatory response (eg cytokines and chemokines) or
bacterial products from the periodontitis associated microflora (eg LPS). Perhaps in the long term
informationlikethiswillhelpwithtreatment of periodontitis patients and patients presented with
co-morbiditiessuch as diabetes, atherosclerosis or kidney disease. In addition (see below), it was
not possible tocompleteall the aimsof the proposal soit would be good to assess how periodontal
neutrophils behave to standard chemoattractants.
Any departures from the original proposal:
In the proposal it was stated that healthy and periodontitis patient neutrophils would be assessed
for chemotaxisagainststandard chemoattractants. Unfortunately the study from which these cells
were tobe takencouldnotstart inthe summer and will now start in the autumn as there were few
patientscomingthroughthe clinicoverthe summer.Howeverthisallowed extra time to look at the
data generated from the second half of the aims.
The value of the studentship:
Throughoutthe 6-weekplacementmyconfidence toworkina lab has increased.Ihave improvedon
labskillssuchas pipetting,dilutingand keeping an aseptic environment, but I have also developed
newskillssuchasmicroscopical techniques in accessing chemotactic properties of neutrophils and
neutrophil extractionandisolationwhich I can to apply in the future if working in a haematological
department.WhatIhave learnthugelyisthatevenwithinaprofessionalstudy there is always room
for adjustment and that sometimes theory remains far from the outcome.
For the lab, a significant piece of data has been generated and analysed by Anice. The data will be
takenfurtherto supportfuture workandhopefullypublications.Asfaras we know no one has done
this type of study before for periodontitis and we are excited by the results. I think that Anice has
gainedmanyskillsandreallyenjoyedhistime interactingwithboththe scientists and clinicians who
make up our group.