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Effect of adipokine priming upon neutrophil oxygen radical and extracellular trap release in response to stimulation by periodontal bacteria and Effect of different adipokine concentrations upon neutrophil directional chemotaxis

Roberts, Helen (2012)
M.Res. thesis, University of Birmingham.

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Project 1:
Obesity is a major public health problem worldwide of increasing prevalence. It is characterised by a low grade chronic inflammatory state, which contributes to obesity-associated morbidity. A body of evidence suggests that obesity is associated with periodontitis, and is second only to smoking as a risk factor for periodontal tissue destruction, but underlying biological mechanisms for this association remain unclear. Adipose tissue functions as an endocrine organ, secreting cytokines (adipokines) that are reported to be associated with dysregulated/inflammatory immune responses to bacterial challenge. Adipokines include tumour necrosis factor alpha (TNF\(\alpha\)), adiponectin, resistin, leptin and interleukin-6 (IL-6), all of which have been shown to prime/affect neutrophils for a secondary stimulus. Neutrophils are the major cell type involved in periodontal immunity to infectious agents. This study therefore aimed to elucidate neutrophil reactive oxygen species (ROS) and neutrophil extracellular trap (NET) responses to bacterial stimulation in an adipokine rich (obese) verses an adipokine normal (lean) environment. Blood was isolated from healthy male volunteers, with no history of diabetes and with normal body mass indices (BMI’s) (range 20-25). Peripheral blood neutrophils were isolated by Percoll\(^{TM}\) discontinuous gradient centrifugation, primed with TNF\(\alpha\), adiponectin, resistin, leptin or IL-6, alongside control two control stimuli GM-CSF and IL-8, at concentrations reflective of lean and obese individuals. Neutrophils were subsequently stimulated with Fusobacterium nucleatum (ATCC 10953) for Toll-Like receptor (TLR)-4 activation, opsonised Staphyloccocus aureus (NCTC 6571) for Fc\( \gamma\)RII activation or phorbol 12-myristate 13-acetate (PMA) for direct activation via protein kinase C (PKC). Neutrophil ROS were measured by luminol enhanced and chemiluminsecence and NET release was by fluorometric assay of DNA and fluorescence microscopy. Adipokine concentrations representative of those found in obese individuals have been shown in this pilot study to enhance neutrophil ROS and NET generation after exposure to bacterial stimulus. The effects are however stimulus dependent and require longer studies to validate any significant results.
Project 2:
The obesity epidemic is a growing public health problem in all worldwide populations. It is well known that obese individuals exhibit low grade chronic inflammation which exacerbates obesity - associated diseases. The link between obesity and the occurrence and severity of periodontitis is well characterised and obesity constitutes a major risk factor for periodontal tissue destruction, however direct biological evidence is yet to be established. Adipose tissue, once overlooked as simply inert, fat storing cells of the body, has been shown to function in a similar way to endocrine organs through the secretion of cytokines (adipokines) can contribute towards dysregulated immune responses to bacterial infection. The globally secreted cytokines tumour necrosis factor (TNFα) and interleukin - 6 (IL - 6) , as well as adipose - specific adipokines resistin, leptin and adiponectin have been previously shown to prime/affect neutrophils which are the primary cells involved in immunity to periodontal bacteria at sufficiently high concentrations. This study aimed to ascertain the effect of these adipokines on neutrophil chemotaxis at concentrations reflective of adipokine rich (obese) and adipokine normal (lean) individuals. Blood was isolated from healthy male volunteers, with no history of diabetes and with normal body mass indices BMI’s (range 20 - 25). Peripheral blood neutrophils were isolated by Percoll TM discontinuous gradient centrifugation. The chemoattractive properties of these adipokines were investigated using an “Insal l” chamber by exposing the neutrophils to lean/obese concentrations. Neutrophil movement was captured by microscopic imaging. The results revealed significant increase in chemotactic index, speed and velocity when the neutrophils were pre-incubated with co ncentrations of adipokines found in obese patients relative to non-obese levels.

Type of Work:M.Res. thesis.
Supervisor(s):Chapple, Iain L. (Iain Leslie) and Grant, Melissa and Lord, Janet and Tomlinson, Jeremy
School/Faculty:Colleges (2008 onwards) > College of Medical & Dental Sciences
Department:School of Dentistry
Subjects:QR Microbiology
QR180 Immunology
RA0421 Public health. Hygiene. Preventive Medicine
RK Dentistry
Institution:University of Birmingham
ID Code:3765
This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder.
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