PGN was chosen as a marker for detection because it is a major component in the cell wall of most bacteria and therefore an abundant and easily targeted antigen. A chromogenic immunohistochemical (IHC) method was adopted for detection of PGN because it has several advantages over fluorescent reporters when examining atherosclerotic plaque. Most importantly, atherosclerotic plaque can be highly auto-fluorescent, which would require lengthy optimisation to establish optimal ‘antigen to auto-fluorescence’ signal ratio. The efficacy of any selected background quencher can only be assessed at the endpoint of an assay, which can make optimisation timely and costly. Fluorescent in-situ hybridisation (FISH) was considered, however aside from auto-fluorescence, FISH has additional challenges, such as long incubation times at elevated temperatures (>60˚C). At high temperature, specimens can detach from the slide causing loss of valuable sample or the buffer containing the probe can evaporate causing specimens to dry out. By adopting a chromogenic IHC procedure, minimal optimisation was required e.g. a simple limit of detection assay to establish optimal antibody concentration. The assay could then be performed at room temperature in <2 h and results could be observed with a readily available light microscope.
PGN was detected in 90% of the CAP tissue specimens examined here. Dense localisation of PGN was predominantly observed in regions containing large accumulations of structures resembling foam cells. These findings are comparable with observations by Laman et al. (2002) who showed PGN localisation in 93.3%, 61.2% and 83.3% in carotid, coronary and femoral atherosclerotic plaque specimens, respectively. Laman et al. (2002)
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examined atherosclerotic carotid and femoral arteries; from adventitia to intima using antibodies for PGN, collagen and macrophages. PGN was detected mainly intracellular and localised within the adventitia, media and atherosclerotic plaque tissue. Consistent with the finding of this chapter, Laman et al. (2002) reported frequent localisation of PGN in areas of plaque tissue occupied by macrophage foam cells (CD14). In the present investigation, the frequency of PGN localisation was positively correlated with the presences of the ghost structures of foam cells. PGN localised around the foam cell shape, but not within the void left by the cell, which often provided extra definition to the ghost structure of accumulated foam cells that would not ordinarily be seen.
Frequent localisation with foam cell structures could possibly represent interaction between PGN and macrophages, possibly even clearance via PGN phagocytosis. However, this observation is beyond the limitations of the assay performed and the scope of this chapter. In order to establish the identification of cells frequently localised by PGN a more specific selection IHC markers would need to be incorporated. Though it must be noted, many of the features encountered in the plaques, including ghost structures of macrophage foam cells, have distinctive histopathology and were often identifiable. Many investigators have previously identified such features using routine H&E and MTC staining (Cai 2002; Salem et al. 2014). Moreover, where necessary and for clarity, textbook images were included alongside any results figures in this chapter to highlight similarities.
Laman et al. (2002) also characterised the vulnerability of lesions in relation to the PGN presence and determined that lesions with significantly higher presence of PGN also displayed certain histological features of a vulnerable plaque phenotype. It was further established that the presence of a lipid core and patient age were positively correlated with intensity of PGN staining; though only deemed significant after the exclusion of two patients negative for PGN. These observations support the notion that atherosclerotic disease may be exacerbated by the presence of bacteria or their cell wall components.
Through employing western blot analysis Laman et al. (2002) established upregulation of toll-like receptor-2 (TLR-2) in PGN positive coronary artery tissue suggesting PGN may represent an inflammatory stimulus causing intracellular signalling for the transcription of proinflammatory genes. Further to this, Nijhuis et al. (2007) demonstrated the ability of PGN to upregulate monocyte expression of L-selectin (CD62L) and β2-integrin-dependent
binding to ICAM-1 under flow cytometry conditions. Monocyte L-selectin expression is a critical step for monocyte tethering and rolling on activated endothelial cells and is the
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precursor to ICAM-1 attachment and chemotaxis. As upregulation of these membrane proteins can assist monocyte transmigration to the subendothelium, these findings further indicate that PGN localisation in the atherosclerotic lesion my stimulate proinflammatory cytokines for leukocyte recruitment and chemotaxis into the artery/lesion.
Nijhuis et al. (2004) utilised enzyme linked immunosorbent assays (ELISA) to measure immunoglobulin levels against PGN in patients with atherosclerosis. The author established that patients hospitalised with atherosclerosis had lower IgM levels directed against PGN compared to control patients without clinically manifest cardiovascular disease. Interestingly, IgM levels against PGN decreased with increasing mean common carotid intimal thickening. The authors provide several explanations for this observation, most fitting of which proposes the decrease in IgM levels are the consequence of effective binding and removal of PGN from the lesion. This explanation does not seem plausible when considering the extensive localisation and frequency of PGN detection in the plaques examined for this chapter. In addition, PGN is located ubiquitously within all bodily mucosa, hence, it seems unlikely that a decrease in IgM level would be due to the effective clearance of PGN, even just from the lesion alone.
Intimal-medial thickening (IMT) and pathological-IMT are features of early to intermediate atherosclerotic plaque development occurring via lipid deposition and associated inflammatory milieu within the intimal layer of the atherosclerotic vessel; as the plaque matures, IMT becomes more apparent (Finn et al. 2010). With this in mind, there is a possibility that as intimal thickening becomes more pronounced, access to the intimal layers becomes more restricted, thus, PGN located within the plaque becomes less accessible, therefore decreasing the overall IgM levels to PGN. However, this also fails to account for the ubiquitous nature of PGN, to which we are continuously exposed at all mucosa.