Correlative Light-Electron Microscopy detects lipopolysaccharide and its association with fibrin fibres in Parkinson's Disease, Alzheimer's Disease and Type 2 Diabetes Mellitus - PubMed (original) (raw)

Correlative Light-Electron Microscopy detects lipopolysaccharide and its association with fibrin fibres in Parkinson's Disease, Alzheimer's Disease and Type 2 Diabetes Mellitus

Greta M de Waal et al. Sci Rep. 2018.

Abstract

Many chronic diseases, including those classified as cardiovascular, neurodegenerative, or autoimmune, are characterized by persistent inflammation. The origin of this inflammation is mostly unclear, but it is typically mediated by inflammatory biomarkers, such as cytokines, and affected by both environmental and genetic factors. Recently circulating bacterial inflammagens such as lipopolysaccharide (LPS) have been implicated. We used a highly selective mouse monoclonal antibody to detect bacterial LPS in whole blood and/or platelet poor plasma of individuals with Parkinson's Disease, Alzheimer's type dementia, or Type 2 Diabetes Mellitus. Our results showed that staining is significantly enhanced (P < 0.0001) compared to healthy controls. Aberrant blood clots in these patient groups are characterized by amyloid formation as shown by the amyloid-selective stains thioflavin T and Amytracker™ 480 or 680. Correlative Light-Electron Microscopy (CLEM) illustrated that the LPS antibody staining is located in the same places as where amyloid fibrils may be observed. These data are consistent with the Iron Dysregulation and Dormant Microbes (IDDM) hypothesis in which bacterial inflammagens such as LPS are responsible for anomalous blood clotting as part of the aetiology of these chronic inflammatory diseases.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1

Figure 1

Overview of this paper, focusing on systemic inflammation in various inflammatory conditions, the presence of inflammagens such as LPS, and its contribution to hypercoagulation and amyloid formation, along with a list of novel research methods employed.

Figure 2

Figure 2

Overview of optimization of LPS antibody binding, using healthy platelet poor plasma (PPP) samples exposed to LPS.

Figure 3

Figure 3

Typical range of confocal micrographs of platelet poor plasma (PPP) with added thrombin, showing the fluorescence amyloid signal of (A) healthy individuals and (B) Parkinson’s Disease (PD) individuals. Platelet poor plasma (PPP) from each individual was incubated with three specific amyloidogenic fluorescent markers, thioflavin T and Amytracker™ 480 and 680.

Figure 4

Figure 4

Representative confocal micrographs of healthy platelet poor plasma (PPP) with added LPS to show optimization of detection of LPS, by using anti-E.coli LPS antibody and a secondary antibody (1:200 dilution for primary and secondary antibodies). We used five different LPS concentrations, and estimated that 0.5 **μ**gL−1 LPS is the lowest detectable concentration. For clarity, we inverted the micrographs, followed by applying the “find edges” function in ImageJ (FIJI), to show the decreasing fluorescence LPS signal with decreasing concentrations added.

Figure 5

Figure 5

Representative platelet poor plasma (PPP) smears with added anti-E.coli LPS antibody and secondary antibody, from (A) healthy individuals, (B) Parkinson’s Disease (PD) individuals and (C) Type 2 Diabetes (T2D) individuals (1:200 dilution for primary and secondary antibodies).

Figure 6

Figure 6

Boxplot of the distribution of the mean fluorescence intensity, normalised by dividing the mean fluorescence intensity values of the healthy individuals, Parkinson’s Disease (PD) individuals and Type 2 Diabetes (T2D) individuals, by the mean fluorescence intensity values of the corresponding secondary antibody control. In this way, we accounted for non-specific secondary antibody binding; ****P < 0.0001.

Figure 7

Figure 7

Representative whole blood (WB) smears with added anti-E.coli LPS antibody and secondary antibody, from (A) healthy individuals, (B) Parkinson’s Disease (PD) individuals and (C) Alzheimer’s Disease (AD) individuals (1:200 dilution for primary and secondary antibodies).

Figure 8

Figure 8

Representative CLEM and SEM micrographs of (A) stored platelet poor plasma (PPP) from a Type 2 Diabetes (T2D) individual, (B) freshly-collected whole blood (WB) from a Parkinson’s Disease (PD) individual and (C) stored whole blood (WB) from a Parkinson’s Disease (PD) individual (1:200 dilution for primary and secondary antibodies). The fluorescence microscopy modalities used were super-resolution (SR-SIM) for (B) and confocal for (A) and (C).

Figure 9

Figure 9

Representative (A) super-resolution (SR-SIM), (B) SEM and (C) CLEM micrographs of freshly-collected whole blood (WB) from a Parkinson’s Disease (PD) individual (1:200 dilution for primary and secondary antibodies). LPS antibody staining is closely associated with the fibre-like structures. Micrograph C colour was enhanced for publication clarity, by adjusting the vibrancy, brightness and contrast in Adobe Photoshop CS6.

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References

    1. Marshall BJ, Warren JR. Unidentified curved bacilli in the stomach of patients with gastritis and peptic ulceration. Lancet. 1984;1:1311–1315. doi: 10.1016/S0140-6736(84)91816-6. - DOI - PubMed
    1. Marshall BJ. Helicobacter pylori. The American journal of gastroenterology. 1994;89:S116–128. - PubMed
    1. De Chiara G, et al. Infectious agents and neurodegeneration. Molecular neurobiology. 2012;46:614–638. doi: 10.1007/s12035-012-8320-7. - DOI - PMC - PubMed
    1. Itzhaki RF. Herpes simplex virus type 1 and Alzheimer’s disease: increasing evidence for a major role of the virus. Frontiers in aging neuroscience. 2014;6:202. doi: 10.3389/fnagi.2014.00202. - DOI - PMC - PubMed
    1. Itzhaki RF, Wozniak MA. Alzheimer’s disease, the neuroimmune axis, and viral infection. Journal of neuroimmunology. 2004;156:1–2. doi: 10.1016/j.jneuroim.2004.07.006. - DOI - PubMed

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