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Do biodegradable micro-particles activate dendritic cells?

Do biodegradable micro-particles activate dendritic cells?

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This project attempted to determine the mechanism of action of Alum, a common adjuvant used since 1924. Alum is good at generating a B cell response, however it is poor at inducing a cell mediate immune response, as a result new adjuvants are being investigated including PLG. My project attempted to determine the mechanism of action of this adjuvant.
This project attempted to determine the mechanism of action of Alum, a common adjuvant used since 1924. Alum is good at generating a B cell response, however it is poor at inducing a cell mediate immune response, as a result new adjuvants are being investigated including PLG. My project attempted to determine the mechanism of action of this adjuvant.

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Published by: Undergraduate Awards on Sep 01, 2012
Copyright:Attribution Non-commercial

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04/27/2014

 
Do Biodegradable microparticles activate dendritic cells?
Abstract
: (maximum 300 words)
 Alum is the only adjuvant approved for human use by the US FDA, however it has so far shown poor results with regards cell-mediated immunity. Biodegradable microparticles based on poly (lactide-co-glycolide) (PLG) polymers and liposomes are proposed as an alternative to alum as they have a longhistory of safe clinical use.Dendritic cells (DCs) are central to the induction of vaccine-specific immune responses, being the onlycells that can activate naive T cells. Many adjuvants including bacterial toxins (Lavelle et al., 2003) canexert their immunostimulatoryeffects on T cells via the activation of DCs. However, the interactionbetweenmicroparticles and DCs have not yet been studied. The main aim of this research was todetermine the effect of different particles on DC activity. DCs were stimulated with various particlesincluding PLG and Polystyrene. ELISA and western blots were then carried out on order to determine DCactivity.It was found that Alum, a known adjuvant, boosts both IL-1
β
and Caspase-1 activity. PLG andPolystyrene cell lysate samples revealed that these particles had no effect on the production of immatureIL-1
β
. However the supernatant samples revealed that the particulates along with a toll agonist resultedin more IL-1
β
than Lipopolysaccharides (LPS) on its own. Because these particulates do not increasethe production of immature IL-1
β
, they must therefore be influencing the processing of IL-1
β
into itsbiologically active form.Caspase-1 was investigated as a possible site of action for these particulates. It was found in the celllysate samples that particulates resulted in more cleaved p10 Caspase-1, then LPS on its own. However further research is required into the inflammasome a multi-protein complex used to cleaveCaspase-1, as it could be the possible site of action for these particulates and so could have importantimplications for adjuvant research in the future.1
 
Project Description
: (maximum 2000 words) to include introduction; materials and methods; results; discussion;conclusion.
Please use diagrams or photographs where appropriate.
Introduction:
Over the past number of decades there has been a move away from the use of live attenuated and wholekilled cell vaccines. The majority of new vaccines, termed ‘subunit vaccines’ comprise purifiedcomponents of organisms such as proteins or polysaccharides. However, in contrast to ‘traditional’vaccines which comprised killed or attenuated organisms, subunit vaccines are generally poorlyimmunogenic.In order to boost the immune responses induced by these vaccines substances called adjuvants are used.The most widely used adjuvant is Aluminium hydroxide’s (Alum) the adjuvant properties of which werefirst described in 1926. Alum is still in widespread use today and is currently the only adjuvant approvedfor human use by the US FDA.However, while alum is an effective inducer of humoral immunity and Th2 type responses, it is not a potent activator of cell-mediated immunity, which is important for protection against diseases includingtuberculosis and HIV. As a result a number of other adjuvants are under investigation and these include biodegradable microparticles based on poly (lactide-co-glycolide (PLG)) polymers and liposomes.Biodegradable microparticles are proposed as an alternative to alum as they have a history of safe clinicaluse. Furthermore there is evidence that poly (lactide-co-glycolide) microparticles can promote strongcellular immune responses.Despite the extensive literature on alum and microparticles there is still very little known about how thesesystems activate the immune response. It is now accepted that dendritic cells are of vital importance in theinduction of adaptive immunity and adjuvants must activate dendritic cells to be effective. The interactionof molecules such as LPS and peptidoglycan with the receptors (TLRs) on the DC induces increasedexpression of major histo-compatibility complex molecules and costimulatory molecules on the cellsurface. These interact with the T cell receptor and ligands for costimulatory molecules on the T cellsurface providing signals 1 and 2 to the cell and supporting T cell proliferation. In addition, on activation,dendritic cells also produce cytokines, which activate and dictate the nature of T cell polarisation towards particular T cell subtypes. While this is well established for lipopolysaccharide (LPS), the nature of theinteraction between particulates such as biodegradable microparticles and dendritic cells is largelyunknown.
2
 
Data from Dr. Ed Lavelle`s laboratory and a recent publication has shown that Alum can promote IL-1 production in dendritic cells (but inhibits IL-12). The paper by Li
et al 
; additionally demonstrated that“Aluminium Hydroxide adjuvants activate Caspase-1 and Induce IL-1
β
and IL-18 release”. This current project sets out to determine if particulates can promote cytokine production by dendritic cells. A particular focus was placed on IL-1 since it is an important proinflammatory cytokine that can drive T cellresponses including ThIL-17 cell induction. Although research into Alum adjuvants has continued theinteraction of micro-particles with dendritic cells has not yet been studied.ELISA data was utilised during this research in order to quantitatively determine the amounts of cytokines produced by dendritic cells. From this we have shown that microparticles induce an increased amount of IL-1
β
, however the mechanism by which this is achieved remains unclear. It is important to note thatmicroparticles on their own do not stimulate IL-1
β
. However in the presence of LPS an increase in IL-1
β
is observed which suggests that microparticles need a toll agonist in order to induce an effect.Caspase-1 (p45) has a molecular weight of 45kDa in its unprocessed form but in its active form it iscleaved into two subunits each with a molecular weight of p10 and p20. Using cell lysate and supernatantssamples the presence of the active form (p10 and p20) was investigated. If large amounts of pro-caspase-1(p10 and p20) are found in the samples, which have been stimulated with microparticles, this mightsuggest that the microparticles are acting on Caspase-1, which would result in large amounts of IL-1
β
 being secreted from the cell.It is hoped that a greater understanding of how particulate vaccine adjuvants promote innate and adaptiveimmunity will help in the design of enhanced vaccine delivery strategies
Materials and Methods:ELISA - Enzyme Linked Immunosorbent Assay:
An important immunological technique used was Enzyme Linked Immunosorbent Assay (ELISA).ELISA is a procedure used to detect small amounts of specific proteins. The assay is based on the bindingof antigens to antibodies to form specific antigen-antibody complexes. These complexes can subsequently be detected by the catalytic action of an enzyme chemically linked to the antibody. The activity of theenzyme is used to determine the level of the substrate present and therefore the amount of antibody present.
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