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- No effects without causes: the Iron Dysregulation and Dormant Microbes hypothesis for chronic, inflammatory diseases
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Whenever authors decide to include references within the text, they need to have mercy to facilitate it for the reader (Me, 2019; Myself, 2019; Irene, 2019), making these less interfering with readability.
"A very large number of chronic, degenerative diseases are accompanied by inflammation. Many of these diseases are extremely common in the modern ‘developed’ world, and include vascular (e.g. atherosclerosis, type 2 diabetes, metabolic syndrome, pre-eclampsia, stroke), autoimmune [e.g. rheumatoid arthritis (RA), multiple sclerosis], and neurodegenerative (e.g. Alzheimer’s, Parkinson’s, Amyotrophic lateral sclerosis) diseases. On the face of it these diseases are quite different from each other, but in fact they share a great many hallmarks [and often comorbidities (see e.g. Agustí & Faner, 2012; Altamura & Muckenthaler, 2009; Figueira et al., 2016; Lago et al., 2011; Nanhoe-Mahabier et al., 2009; Pretorius, Mbotwe & Kell, 2017b; Shen et al., 2016)]. As well as inflammation, these [disease] hallmarks include increased levels of inflammatory cytokines (almost a definition of inflammation), dysregulation in iron metabolism [especially the appearance of abnormal levels of ferritin in the serum (Kell & Pretorius, 2014)], and a variety of coagulopathies and haematological pathologies (abnormalities in the blood system, including its clotting properties). Many of these diseases also share other properties such as an increase in ‘insoluble’ forms of normally soluble proteins and of microparticulate material. Although they are progressive diseases, their progress is far from uniform, and they are often accompanied by fluctuating changes in physiological states (such as ‘flares’ in rheumatoid arthritis)."
"However, these ‘hallmarks’ are effectively physiological biomarkers; they are responses to one or more initial external stimuli, and they can and do serve as mediators for (later) manifestations of overt disease. Since effects do not happen without causes, however, the question then arises as to the identity of these external stimuli. In some cases (especially atherosclerosis and metabolic syndrome) there is evidence for a significant dietary component. However, based on a now considerable and wide-ranging literature, we here bring together evidence that: (i) the main external stimuli are microorganisms; (ii) in contrast to what happens in conventional infectious diseases they do not proliferate unchecked, but commonly enter dormant states that make them invisible to classical microbiology; and (iii) they can be reactivated from these dormant states by the presence of ‘free’ iron (a necessary nutrient that in unliganded form is normally at low levels in the host). This reactivation releases highly potent inflammagens such as lipopolysaccharide (LPS) from Gram-negative organisms and lipoteichoic acid (LTA) from Gram-positives. Various sequelae, including coagulopathies, amyloid formation and cell death follow from this, and thus we argue that this general explanation – that we refer to here as the Iron Dysegulation and Dormant Microbes (IDDM) hypothesis– underpins a host of these chronic, inflammatory diseases."
"
"However, these ‘hallmarks’ are effectively physiological biomarkers; they are responses to one or more initial external stimuli, and they can and do serve as mediators for (later) manifestations of overt disease. Since effects do not happen without causes, however, the question then arises as to the identity of these external stimuli. In some cases (especially atherosclerosis and metabolic syndrome) there is evidence for a significant dietary component. However, based on a now considerable and wide-ranging literature, we here bring together evidence that: (i) the main external stimuli are microorganisms; (ii) in contrast to what happens in conventional infectious diseases they do not proliferate unchecked, but commonly enter dormant states that make them invisible to classical microbiology; and (iii) they can be reactivated from these dormant states by the presence of ‘free’ iron (a necessary nutrient that in unliganded form is normally at low levels in the host). This reactivation releases highly potent inflammagens such as lipopolysaccharide (LPS) from Gram-negative organisms and lipoteichoic acid (LTA) from Gram-positives. Various sequelae, including coagulopathies, amyloid formation and cell death follow from this, and thus we argue that this general explanation – that we refer to here as the Iron Dysegulation and Dormant Microbes (IDDM) hypothesis– underpins a host of these chronic, inflammatory diseases."
"
- A systems biology strategy was used to show that chronic, inflammatory diseases have many features in common besides simple inflammation.
- The physiological state of most microbes in nature is neither ‘alive’ (immediately culturable on media known to support their growth) nor ‘dead’ (incapable of such replication), but dormant.
- The inflammatory features of chronic diseases must have external causes, and we suggest that the chief external causes are (i) inoculation by microbes that become and remain dormant, largely because they lack the free iron necessary to replicate, and (ii) traumas that induce cell death and the consequent liberation of free iron; these together are sufficient to initiate replication of the microbes.
- This replication is accompanied by the production and shedding of potent inflammagens such as lipopolysaccharide or lipoteichoic acid, and this continuing release explains the presence of chronic, low-grade inflammation.
- Recent findings show that tiny amounts of these inflammagens can cause blood to clot into an amyloid form; such amyloid forms are also capable of inducing cell death and thereby exacerbating the release of iron.
- Additional to the formal literature that we have reviewed here, it seems to be commonly known that infection is in fact the proximal cause of death in Alzheimer’s, Parkinson’s, rheumatoid arthritis, multiple sclerosis, etc. It may, for instance, be brought on by the trauma experienced following a fall. Such infections leading to death in chronically ill patients may involve the re-awakening of dormant bacteria rather than novel exogenous infection. This implies that therapies involving the careful use of anti-infectives active against dormant microbes could be effective (Coates, Halls & Hu, 2011; Coates & Hu, 2006), as well as the use of nutritional iron chelators (Kell, 2009; Perron & Brumaghim, 2009; Perron et al., 2010).
- The role of microbes in stomach ulcers is now well established (Marshall, 2002a,b, 2003, 2006); here we add to the list of supposedly non-communicable diseases that can be shown to have a microbial component in their aetiology.
"
"While microbiomes such as the skin microbiome (Dréno et al., 2016; Dybboe et al., 2017; Edmonds-Wilson et al., 2015; Fitz-Gibbon et al., 2013; Kong et al., 2017; Kong et al., 2012; Oh et al., 2016, 2013; SanMiguel & Grice, 2015; van Rensburg et al., 2015) and the gut microbiome (see Section II.1) are well known, many other sites that are widely considered sterile are in fact full of microbes (Bullman, Meyerson & Kostic, 2017; Ding & Schloss, 2014; Foster et al., 2017; Garn et al., 2016; The Human Microbiome Project Consortium, 2012; Lloyd & Marsland, 2017; Lluch et al., 2015). As well as blood, which we also discuss in detail herein, these include the respiratory system (e.g. Bassis et al., 2015; Budden et al., 2017; Dickson et al., 2017, 2016, b; Dickson & Huffnagle, 2015; Huffnagle, Dickson & Lukacs, 2017; O’Dwyer, Dickson & Moore, 2016; Samuelson, Welsh & Shellito, 2015; Vientós-Plotts et al., 2017, b), neck tissue (Wang et al., 2017), breast tissue (Wang et al., 2017), and both seminal fluid (Craig et al., 2015; Hou et al., 2013; Javurek et al., 2016; Kenny & Kell, 2018; C.M. Liu et al., 2014; Mändar et al., 2015; Weng et al., 2014) and the placenta (Aagaard et al., 2014; Amarasekara et al., 2015; Antony et al., 2015; Collado et al., 2016; Pelzer et al., 2016; Prince et al., 2016; Tarazi, Agostoni & Kim, 2014; Zheng et al., 2015) (cf . Lauder et al., 2016). Indeed, probably all tissues harbour fairly considerable numbers of non-growing microbes even under normal conditions (Bullman et al., 2017; Domingue, Turner & Schlegel, 1974; Domingue, 2010; Domingue & Woody, 1997; Gargano & Hughes, 2014; Mattman, 2001; Proal, Albert & Marshall, 2013, 2014; Proal, Lindseth & Marshall, 2017)."
"Almost everything dietary, including medicines (Le Bastard et al., 2017), can affect the gut microbiome [and vice versa (Gillis et al., 2018; Koppel, Maini Rekdal & Balskus, 2017; Wilson & Nicholson, 2017)], and there is a large literature, that we do not seek to summarise (Subramanian et al., 2015), on the use of prebiotics and probiotics that are intended to modify it. There is consequently no such thing as a or the ‘normal’ gut microbiome, although certain patterns or frequencies of microbial types are seen as representing some kind of commonality (Lloyd-Price et al., 2017), at least to the ethnic group under study. For our purposes, the main significance is that the gut microbiome is large and that it exists. ‘Dysbiosis’ is a term usually used to mean a change in the gut microbiome such that its composition differs significantly from those of the ‘normal’ (commonest) populations of interest (Olesen & Alm, 2016) and we adopt this usage herein. Unfortunately, ‘dysbiosis’ is also used, misleadingly, to refer to the appearance of gut microbes in other places; we have therefore suggested the use of the word ‘atopobiosis’ for this latter meaning [microbes in the ‘wrong’ place (Potgieter et al., 2015)]."
"Large amounts of insoluble LPS are [] present in the gut (∼1 g; Zaman & Zaman, 2015), and these too can pass into the bloodstream (de Punder & Pruimboom, 2015; Kell & Pretorius, 2015a; Maes, Coucke & Leunis, 2007)."
"A second common origin for blood microbes is the non-sterile oral cavity (Gargano & Hughes, 2014), whence they can enter through abrasive toothbrushing (Bhanji et al., 2002; Tomás et al., 2012) or periodontal disease. Since blood can appear in the oral cavity, there is nothing to stop the reverse process of microbial infection of the blood (Dhotre, Davane & Nagoba, 2017; Kilian et al., 2016; Koren et al., 2011) and periodontal origins represent another source of potential microbial translocation (Moon & Lee, 2016). There is considerable evidence for a significant association between periodontitis and RA (Bingham III & Moni, 2013; Cheng et al., 2018; de Smit et al., 2012; Detert et al., 2010; Konig et al., 2016; Koziel, Mydel & Potempa, 2014; Lee et al., 2015; Martinez-Martinez et al., 2009; Mikuls et al., 2009; Monsarrat et al., 2013; Ogrendik, 2013; Potempa, Mydel & Koziel, 2017). Atherosclerosis provides another example (Chukkapalli et al., 2015; Gibson III & Genco, 2007; Kebschull, Demmer & Papapanou, 2010; Łysek et al., 2017; Mahalakshmi et al., 2017; Rangé et al., 2014; Reyes et al., 2013; Rivera et al., 2013; Teeuw et al., 2014; Toyofuku et al., 2011; Velsko et al., 2014)."
"The chief method of classical microbiology involves plating a suitably diluted subsample from the sample of interest onto a ‘solid’ (usually agar) medium considered likely to allow their proliferation, and waiting until visible colonies are formed, the number of ‘colony-forming units’ (CFUs) being equal to the number of ‘viable’ bacteria in the subsample. There are numerous growth media [the classic listing (Zimbro et al., 2009) runs to 700 pages], and typically rather ‘rich’ media are used. One such medium, known euphemistically as ‘chocolate’ agar, is based on blood that has been heated to 80◦C to lyse erythrocytes. The concept that ‘viability’ = culturability, or the ability to replicate, is thus a cornerstone of microbiology (Postgate, 1967, 1969, 1976)."
"The problem with this general strategy is that not only are individual media not suitable for all organisms, but that most organisms (especially when starved) can enter physiological states in which rich media either do not support their growth or may actually kill them (and clearly it is hard to discriminate between these possibilities). However, the organisms may not be ‘dead’, as other treatments can restore them to a physiological state in which they do produce colonies on the same media. Under these circumstances we should refer to them as ‘dormant’ (Kaprelyants, Gottschal & Kell, 1993) since clearly they are not ‘dead’ – a state we take on classical semantic grounds to be irreversible."
"Were the microbes that enter the blood to be capable of replicating in a medium that – like ‘chocolate’ agar – is actually quite rich in organic molecules, we would be discussing conventional, infectious diseases and bacteraemia as commonly understood, but we are not. Under normal conditions, however, either because of the innate immune system or the physiological state of the microbes, or both, normal (non-bacteraemic) blood – as judged by classical microbiological criteria – is indeed sterile, i.e. it is not possible to detect the presence of viable bacteria in this way. To investigate whether dormant bacteria are present, we thus need culture-independent methods, of which ultramicroscopic (e.g. Domingue et al., 1974; Domingue, 1995, 2010; Domingue&Woody, 1997; Ewald, 2002; Green, Heidger Jr & Domingue, 1974a,b; Mattman, 2001; Potgieter et al., 2015) and molecular sequence-based methods (Amar et al., 2011; Cherkaoui et al., 2009; Fern´andez-Cruz et al., 2013; Gaibani et al., 2013; Grif et al., 2012,b; C.L. Liu et al., 2014; Moriyama et al., 2008; NIH HMP Working Group et al., 2009; Nikkari et al., 2001; Sakka et al., 2009; Sato et al., 2014; Valencia-Shelton & Loeffelholz, 2014; Woyke, Doud & Schulz, 2017) are by far the most common."
"We also recognise that dormant bacteria can survive in white blood cells (Liehl, Zuzarte-Luis & Mota, 2015; Miskinyte & Gordo, 2013; Miskinyte et al., 2013; Ribet & Cossart, 2015; Thwaites & Gant, 2011), and probably also in the much more prevalent red blood cells (Potgieter et al., 2015), just as can classically infectious organisms such as Bartonella spp. (Ben-Tekaya, Gorvel&Dehio, 2013; Dehio, 2001; Eicher & Dehio, 2012; Pitassi et al., 2007; Seubert, Schulein & Dehio, 2002), Francisella tularensis (Conlan, 2011; Horzempa et al., 2011), various mycoplasmas (e.g. Groebel et al., 2009), and Streptococcus pneumoniae (Yamaguchi et al., 2013)."
"[..]those recognising relationships between overt chronic, inflammatory disease and the presence of detectable microbes, can highlight that the blood and tissue microbiome is greatly enhanced in these diseases (Alonso et al., 2017; Arleevskaya et al., 2016; Berstad & Berstad, 2017; Broxmeyer, 2017a,b; Ebringer, 2012; Ebringer & Rashid, 2009; Ebringer, Rashid & Wilson, 2010; Emery et al., 2017; Itzhaki et al., 2016; Kell & Kenny, 2016; Maheshwari & Eslick, 2015; Miklossy, 2011; Miklossy &McGeer, 2016; Pisa et al., 2017; Pretorius et al., 2017a; Pretorius, Bester & Kell, 2016a; Proal et al., 2013, 2014, 2017). We note too that while it is all too easy to dismiss such findings as ‘contaminants’, those doing so must also explain why the microbes appear at much higher levels only in the ‘disease’ samples."
"It has been pointed out previously (e.g. Mangin, Sinha & Fincher, 2014; Proal, Albert & Marshall, 2015) that vitamin D dysregulation is a common accompaniment to chronic infection with (normally) dormant microbes. Vitamin D dysregulation typically manifests as a low serum level of calcidiol [25-hydroxy-D3; 25(OH)D3] and is indeed widely observed in inflammation (Table 1), although whether it is a cause or a consequence cannot of course be determined from simple co-occurrences. The studies listed in Table 1 show associations, but not (Beveridge & Witham, 2013; Cannell, Grant & Holick, 2014; Kienreich et al., 2013) whether low vitamin D levels are a cause or an effect of inflammation (or both, under different conditions; Cannell et al., 2014), how this relates to the disease, and whether improving some aspect of vitamin D status would be a treatment option."
"[..]iron dysregulation can be initiated by a multitude of factors that cause cell death, which will release free iron into the bloodstream, whence it can be disseminated throughout the body (Kell & Pretorius, 2014). Such factors include mechanical damage [including trauma (Gorbunov et al., 2006, 2005, 2003; Zhang et al., 2013) and dysbiosis], nutritional stress (Schaffer, 2003, 2016), pharmacological stress (Pirmohamed et al., 2004), oxidative stress (Crichton, 2016; Kerley et al., 2018) and others (Nanba et al., 2016), many of which also involve the production of stress hormones."
"In contrast to apoptosis in nucleated cells, programmed cell death in red blood cells (RBCs) is known as eryptosis (Bissinger et al., 2013; Föller et al., 2008; Lang & Lang, 2015; E. Lang, Qadri & Lang, 2012a; Lang et al., 2010; F. Lang, Lang & Foller, 2012b; Lang & Qadri, 2012; Pretorius, du Plooy & Bester, 2016b; Qadri et al., 2011; Qadri et al., 2016; Qadri et al., 2012). It causes the release of haem from RBCs, which can eventually lead to the presence of free ‘iron’. The physiological processes taking place during eryptosis are similar to those of apoptosis, but without the involvement of the nucleus and mitochondria."
"‘Chocolate’ agar is a medium widely used for assaying bacteria via their growth, and is essentially heated blood. However, bacteria proliferate much less well in actual blood, partly due to the presence of antimicrobial components and the innate immune system but also because healthy blood in vivo normally has almost no free iron available (1–10 μM) (Armitage & Drakesmith, 2014; Chu et al., 2010; Haley & Skaar, 2012; Sivick & Mobley, 2010; Subashchandrabose & Mobley, 2015; Wessling-Resnick, 2010). Indeed iron-withholding (Ganz, 2009; Jurado, 1997; Nevitt, 2011; Weinberg, 2009; Weinberg & Miklossy, 2008) is a major strategy used by hosts to inhibit the growth of microbial invaders. This is often described as a ‘battle’ (Armitage & Drakesmith, 2014; Carver, 2018; Chu et al., 2010; Damron et al., 2016; Fischbach et al., 2006; Haley & Skaar, 2012; Pich & Merrell, 2013; Skaar, 2010; Stijlemans et al., 2015) or ‘struggle’ (Markel et al., 2007; Nairz et al., 2010; Reid, Anderson & Lamont, 2009) for iron between the host and invader."
"Although we suspect that the greater significance of free iron in chronic, inflammatory diseases is via microbial activation (Fig. 1) rather than via the Fenton and Haber–Weiss reactions and oxidative stress, there is no doubt that excess iron is itself directly involved in a variety of diseases (Table 2)."
"The cell walls of Gram-negative and Gram-positive bacteria contain significant amounts of LPS and LTA that can become detached in response to different environmental and physiological signals (e.g. Watson et al., 1977). When shed into the host, LPS is known as endotoxin. The most extreme example of microbial shedding of inflammatory material of this type is in a condition known as the Jarisch–Herxheimer reaction (Almeida, Estanqueiro & Salgado, 2016; Belum et al., 2013; Cheung & Chee, 2009; Guerrier & D’Ortenzio, 2013; Kadam et al., 2015; Pound & May, 2005; See, Scott & Levin, 2005), which is essentially an uncontrolled cytokine storm (see Section X) caused by the rapid release of inflammagenic cell wall materials from microbes, often following bactericidal antibiotic treatment (Lepper et al., 2002)."
"The inflammagenic potency of LPS is so great that it is commonly (and ironically) even used as a model to induce symptoms more or less similar to many of the inflammatory diseases of interest. Typically this involves injecting LPS at the site of interest for such diseases. Examples of the use of endotoxin in this way include pre-eclampsia (Cotechini et al., 2014; Faas et al., 1994; Faas et al., 2000; Lin et al., 2012; Liu et al., 2017; Rademacher, Gumaa & Scioscia, 2007; Sakawi et al., 2000; Williamson et al., 2016; Xue et al., 2015), Alzheimer’s (Zhan et al., 2015, 2016), Parkinson’s (Barnum & Tansey, 2010; Byler et al., 2009; Cunningham et al., 2005; He et al., 2013; Hoban et al., 2013; Hritcu & Ciobica, 2013; Hritcu et al., 2011; Liu&Bing, 2011; Miller et al., 2009; Orr, Rowe & Halliday, 2002; Santiago et al., 2010; Tufekci, Genc & Genc, 2011; Z. Zhang et al., 2012), rheumatoid arthritis (Izui, Eisenberg & Dixon, 1979; Nemeth et al., 1985), atherosclerosis (Khedoe et al., 2013), multiple sclerosis (di Penta et al., 2013; Nguyen et al., 2004), Guillain-Barré syndrome (Prendergast &Moran, 2000), sepsis (Lewis, Seymour & Rosengart, 2016; Remick & Ward, 2005), and stroke (Becker et al., 2005; Doll et al., 2015; Shim & Wong, 2016). This far-from-exhaustive list illustrates well the generality of this phenomenon. In cases of stroke, infection is very common, and leads to a worse prognosis; in some cases antibiotics worsen it further (Becker et al., 2016), consistent with the view that the infecting organisms were already present, and that there is an active role of LPS shedding. We note too that some molecules such as P-type inositol phosphate glycans can act as LPS mimics (Robillard et al., 2016). This is especially well established in pre-eclampsia (e.g. Dawonauth et al., 2014; Kenny & Kell, 2018; Robillard et al., 2016; Scioscia et al., 2012, 2011; Williams et al., 2007) but seems to have been little investigated elsewhere."
"Consistent with the above (and see de Punder & Pruimboom, 2015; Kell & Pretorius, 2015a), Table 3 lists a variety of ‘natural’ (i.e. non-experimental) chronic inflammatory diseases for which it has been shown that steady-state endotoxin (LPS) levels are raised and Table 4 presents examples of diseases in which raised levels of lipopolysaccharide binding protein (LBP) have been observed."
"Gram-positive bacteria have a cell wall structure that differs from that of Gram-negatives both in its number of barriers and in the fact that the cell wall component equivalent to LPS is lipoteichoic acid (LTA). LTA is equivalently capable of producing an inflammatory response. In contrast to LPS, which mainly interacts with toll-like receptor 4 (TLR4) (Balasubbramanian et al., 2017; Hoshino et al., 1999; Kell & Pretorius, 2015a; Lien et al., 2000; Poltorak et al., 1998), LTA stimulates target cells mainly by activating toll-like receptor 2 (TLR2) (Ishii & Akira, 2004; Jiménez-Dalmaroni, Gerswhin & Adamopoulos, 2016; Kawai & Akira, 2011; Kumar, Kawai & Akira, 2011; Kumar et al., 2013; Y. Liu et al., 2014; Mukherjee, Karmakar & Babu, 2016; Oliveira-Nascimento, Massari &Wetzler, 2012; Schwandner et al., 1999; Underhill et al., 1999; Zähringer et al., 2008). The glycolipid anchor of LTA plays a central role, analogous to lipid A of LPS (Morath, von Aulock & Hartung, 2005)."
"LTA species have been rather less studied from the point of view of inflammagenesis than have LPS forms, but they clearly reside in the blood and are inflammagens (Barbero-Becerra et al., 2011; Cinar et al., 2013; Hoogerwerf et al., 2009; Levels et al., 2003; Pirillo, Catapano & Norata, 2015). In some respects (see Section X), LTAs may be even more potent than LPS species (Pretorius et al., 2018a)."
"A variety of small-molecule (Donia & Fischbach, 2015) microbial products besides LPS and LTA, such as long- (Schirmer et al., 2016) and short-chain (Thorburn, Macia & Mackay, 2014) fatty acids, can also lead to or modulate the formation of inflammatory cytokines."
"‘Amyloid’, more specifically an amyloid protein fibril, is defined formally (Sipe et al., 2014, p. 221) as ‘a protein that is deposited as insoluble fibrils, mainly in the extracellular spaces of organs and tissues as a result of sequential changes in protein folding that result in a condition known as amyloidosis’. As with prions (Aguzzi & Lakkaraju, 2016; Kell & Pretorius, 2017a; Prusiner, 1998; Prusiner et al., 2015), there is (or need be) no change in the primary sequence when a normally soluble protein adopts an insoluble amyloid form. Anfinsen’s (1973) classical experiments had implied that the primary sequence alone can be sufficient to guide normal folding and that folding was to the state of lowest free energy. The existence of more stable conformations than those first formed upon folding implies, in contrast to this, that there is a large kinetic barrier between the most common conformation and the folded amyloid form(s) of lower free energy (Cohen & Prusiner, 1998) (Fig. 6). As many as 50 ‘amyloid’ diseases are now established (Ankarcrona et al., 2016; Buell, Dobson & Knowles, 2014; Dobson, 2013; Hung et al., 2016; Ke et al., 2017; Kholová & Niessen, 2005; Knowles, Vendruscolo & Dobson, 2014; Siakallis, Tziakouri-Shiakalli & Georgiades, 2014), in which normally soluble proteins fold to form unusual, insoluble amyloid fibril forms and may become on and off-pathway oligomers that are particularly important for cytotoxicity (Ke et al., 2017)."
"Even proteins not normally seen as amyloidogenic or disease-causing can form amyloids; this is of significance in the storage of biological materials, whose shelf-life may be shortened as a result [e.g. insulin (Nielsen et al., 2001,b,c; Wang, 2005)]."
"[..]amyloidogenesis could be induced to occur by the addition of what is stoichiometrically an astonishingly low ratio of bacterial lipopolysaccharide (LPS):fibrinogen, 1:10^8."
"As well as its role in inducing inflammatory cytokine production, there is some evidence that LPS, albeit commonly bound to proteins that can sequester it, is itself directly cytotoxic [reviewed by Kell & Pretorius (2015a) and Williamson et al. (2016)]."
"A general feature of the blood of patients with these chronic inflammatory diseases is that it is both hypercoagulable and hypofibrinolytic (Kell & Pretorius, 2015b); clots form more easily, are stronger, and are less susceptible to proteolysis. The latter is, of course, a particular hallmark of prions (Basu et al., 2007; Saá & Cervenakova, 2015; Saleem et al., 2014; Silva et al., 2015; Woerman et al., 2018) and of amyloids generally (Rambaran & Serpell, 2008)."
"It is hard to disentangle diseases caused or exacerbated directly by inflammation from those where the mediating agent is explicitly a cytokine."
"The different steps considered herein are entirely generic at a broad level (microbes and their dormant states, iron dysregulation, amyloid formation), with differences only apparent at a finer scale (microbial species and the anatomical location of the various dysregulations). The conditions considered herein are all chronic inflammatory diseases, often with quite slow kinetics, and are all in effect diseases of ageing (e.g. van Beek, Kirkwood & Bassingthwaighte, 2016)."
"Almost everything dietary, including medicines (Le Bastard et al., 2017), can affect the gut microbiome [and vice versa (Gillis et al., 2018; Koppel, Maini Rekdal & Balskus, 2017; Wilson & Nicholson, 2017)], and there is a large literature, that we do not seek to summarise (Subramanian et al., 2015), on the use of prebiotics and probiotics that are intended to modify it. There is consequently no such thing as a or the ‘normal’ gut microbiome, although certain patterns or frequencies of microbial types are seen as representing some kind of commonality (Lloyd-Price et al., 2017), at least to the ethnic group under study. For our purposes, the main significance is that the gut microbiome is large and that it exists. ‘Dysbiosis’ is a term usually used to mean a change in the gut microbiome such that its composition differs significantly from those of the ‘normal’ (commonest) populations of interest (Olesen & Alm, 2016) and we adopt this usage herein. Unfortunately, ‘dysbiosis’ is also used, misleadingly, to refer to the appearance of gut microbes in other places; we have therefore suggested the use of the word ‘atopobiosis’ for this latter meaning [microbes in the ‘wrong’ place (Potgieter et al., 2015)]."
"Large amounts of insoluble LPS are [] present in the gut (∼1 g; Zaman & Zaman, 2015), and these too can pass into the bloodstream (de Punder & Pruimboom, 2015; Kell & Pretorius, 2015a; Maes, Coucke & Leunis, 2007)."
"A second common origin for blood microbes is the non-sterile oral cavity (Gargano & Hughes, 2014), whence they can enter through abrasive toothbrushing (Bhanji et al., 2002; Tomás et al., 2012) or periodontal disease. Since blood can appear in the oral cavity, there is nothing to stop the reverse process of microbial infection of the blood (Dhotre, Davane & Nagoba, 2017; Kilian et al., 2016; Koren et al., 2011) and periodontal origins represent another source of potential microbial translocation (Moon & Lee, 2016). There is considerable evidence for a significant association between periodontitis and RA (Bingham III & Moni, 2013; Cheng et al., 2018; de Smit et al., 2012; Detert et al., 2010; Konig et al., 2016; Koziel, Mydel & Potempa, 2014; Lee et al., 2015; Martinez-Martinez et al., 2009; Mikuls et al., 2009; Monsarrat et al., 2013; Ogrendik, 2013; Potempa, Mydel & Koziel, 2017). Atherosclerosis provides another example (Chukkapalli et al., 2015; Gibson III & Genco, 2007; Kebschull, Demmer & Papapanou, 2010; Łysek et al., 2017; Mahalakshmi et al., 2017; Rangé et al., 2014; Reyes et al., 2013; Rivera et al., 2013; Teeuw et al., 2014; Toyofuku et al., 2011; Velsko et al., 2014)."
"The chief method of classical microbiology involves plating a suitably diluted subsample from the sample of interest onto a ‘solid’ (usually agar) medium considered likely to allow their proliferation, and waiting until visible colonies are formed, the number of ‘colony-forming units’ (CFUs) being equal to the number of ‘viable’ bacteria in the subsample. There are numerous growth media [the classic listing (Zimbro et al., 2009) runs to 700 pages], and typically rather ‘rich’ media are used. One such medium, known euphemistically as ‘chocolate’ agar, is based on blood that has been heated to 80◦C to lyse erythrocytes. The concept that ‘viability’ = culturability, or the ability to replicate, is thus a cornerstone of microbiology (Postgate, 1967, 1969, 1976)."
"The problem with this general strategy is that not only are individual media not suitable for all organisms, but that most organisms (especially when starved) can enter physiological states in which rich media either do not support their growth or may actually kill them (and clearly it is hard to discriminate between these possibilities). However, the organisms may not be ‘dead’, as other treatments can restore them to a physiological state in which they do produce colonies on the same media. Under these circumstances we should refer to them as ‘dormant’ (Kaprelyants, Gottschal & Kell, 1993) since clearly they are not ‘dead’ – a state we take on classical semantic grounds to be irreversible."
"Were the microbes that enter the blood to be capable of replicating in a medium that – like ‘chocolate’ agar – is actually quite rich in organic molecules, we would be discussing conventional, infectious diseases and bacteraemia as commonly understood, but we are not. Under normal conditions, however, either because of the innate immune system or the physiological state of the microbes, or both, normal (non-bacteraemic) blood – as judged by classical microbiological criteria – is indeed sterile, i.e. it is not possible to detect the presence of viable bacteria in this way. To investigate whether dormant bacteria are present, we thus need culture-independent methods, of which ultramicroscopic (e.g. Domingue et al., 1974; Domingue, 1995, 2010; Domingue&Woody, 1997; Ewald, 2002; Green, Heidger Jr & Domingue, 1974a,b; Mattman, 2001; Potgieter et al., 2015) and molecular sequence-based methods (Amar et al., 2011; Cherkaoui et al., 2009; Fern´andez-Cruz et al., 2013; Gaibani et al., 2013; Grif et al., 2012,b; C.L. Liu et al., 2014; Moriyama et al., 2008; NIH HMP Working Group et al., 2009; Nikkari et al., 2001; Sakka et al., 2009; Sato et al., 2014; Valencia-Shelton & Loeffelholz, 2014; Woyke, Doud & Schulz, 2017) are by far the most common."
"We also recognise that dormant bacteria can survive in white blood cells (Liehl, Zuzarte-Luis & Mota, 2015; Miskinyte & Gordo, 2013; Miskinyte et al., 2013; Ribet & Cossart, 2015; Thwaites & Gant, 2011), and probably also in the much more prevalent red blood cells (Potgieter et al., 2015), just as can classically infectious organisms such as Bartonella spp. (Ben-Tekaya, Gorvel&Dehio, 2013; Dehio, 2001; Eicher & Dehio, 2012; Pitassi et al., 2007; Seubert, Schulein & Dehio, 2002), Francisella tularensis (Conlan, 2011; Horzempa et al., 2011), various mycoplasmas (e.g. Groebel et al., 2009), and Streptococcus pneumoniae (Yamaguchi et al., 2013)."
"[..]those recognising relationships between overt chronic, inflammatory disease and the presence of detectable microbes, can highlight that the blood and tissue microbiome is greatly enhanced in these diseases (Alonso et al., 2017; Arleevskaya et al., 2016; Berstad & Berstad, 2017; Broxmeyer, 2017a,b; Ebringer, 2012; Ebringer & Rashid, 2009; Ebringer, Rashid & Wilson, 2010; Emery et al., 2017; Itzhaki et al., 2016; Kell & Kenny, 2016; Maheshwari & Eslick, 2015; Miklossy, 2011; Miklossy &McGeer, 2016; Pisa et al., 2017; Pretorius et al., 2017a; Pretorius, Bester & Kell, 2016a; Proal et al., 2013, 2014, 2017). We note too that while it is all too easy to dismiss such findings as ‘contaminants’, those doing so must also explain why the microbes appear at much higher levels only in the ‘disease’ samples."
"It has been pointed out previously (e.g. Mangin, Sinha & Fincher, 2014; Proal, Albert & Marshall, 2015) that vitamin D dysregulation is a common accompaniment to chronic infection with (normally) dormant microbes. Vitamin D dysregulation typically manifests as a low serum level of calcidiol [25-hydroxy-D3; 25(OH)D3] and is indeed widely observed in inflammation (Table 1), although whether it is a cause or a consequence cannot of course be determined from simple co-occurrences. The studies listed in Table 1 show associations, but not (Beveridge & Witham, 2013; Cannell, Grant & Holick, 2014; Kienreich et al., 2013) whether low vitamin D levels are a cause or an effect of inflammation (or both, under different conditions; Cannell et al., 2014), how this relates to the disease, and whether improving some aspect of vitamin D status would be a treatment option."
"[..]iron dysregulation can be initiated by a multitude of factors that cause cell death, which will release free iron into the bloodstream, whence it can be disseminated throughout the body (Kell & Pretorius, 2014). Such factors include mechanical damage [including trauma (Gorbunov et al., 2006, 2005, 2003; Zhang et al., 2013) and dysbiosis], nutritional stress (Schaffer, 2003, 2016), pharmacological stress (Pirmohamed et al., 2004), oxidative stress (Crichton, 2016; Kerley et al., 2018) and others (Nanba et al., 2016), many of which also involve the production of stress hormones."
"In contrast to apoptosis in nucleated cells, programmed cell death in red blood cells (RBCs) is known as eryptosis (Bissinger et al., 2013; Föller et al., 2008; Lang & Lang, 2015; E. Lang, Qadri & Lang, 2012a; Lang et al., 2010; F. Lang, Lang & Foller, 2012b; Lang & Qadri, 2012; Pretorius, du Plooy & Bester, 2016b; Qadri et al., 2011; Qadri et al., 2016; Qadri et al., 2012). It causes the release of haem from RBCs, which can eventually lead to the presence of free ‘iron’. The physiological processes taking place during eryptosis are similar to those of apoptosis, but without the involvement of the nucleus and mitochondria."
"‘Chocolate’ agar is a medium widely used for assaying bacteria via their growth, and is essentially heated blood. However, bacteria proliferate much less well in actual blood, partly due to the presence of antimicrobial components and the innate immune system but also because healthy blood in vivo normally has almost no free iron available (1–10 μM) (Armitage & Drakesmith, 2014; Chu et al., 2010; Haley & Skaar, 2012; Sivick & Mobley, 2010; Subashchandrabose & Mobley, 2015; Wessling-Resnick, 2010). Indeed iron-withholding (Ganz, 2009; Jurado, 1997; Nevitt, 2011; Weinberg, 2009; Weinberg & Miklossy, 2008) is a major strategy used by hosts to inhibit the growth of microbial invaders. This is often described as a ‘battle’ (Armitage & Drakesmith, 2014; Carver, 2018; Chu et al., 2010; Damron et al., 2016; Fischbach et al., 2006; Haley & Skaar, 2012; Pich & Merrell, 2013; Skaar, 2010; Stijlemans et al., 2015) or ‘struggle’ (Markel et al., 2007; Nairz et al., 2010; Reid, Anderson & Lamont, 2009) for iron between the host and invader."
"Although we suspect that the greater significance of free iron in chronic, inflammatory diseases is via microbial activation (Fig. 1) rather than via the Fenton and Haber–Weiss reactions and oxidative stress, there is no doubt that excess iron is itself directly involved in a variety of diseases (Table 2)."
"The cell walls of Gram-negative and Gram-positive bacteria contain significant amounts of LPS and LTA that can become detached in response to different environmental and physiological signals (e.g. Watson et al., 1977). When shed into the host, LPS is known as endotoxin. The most extreme example of microbial shedding of inflammatory material of this type is in a condition known as the Jarisch–Herxheimer reaction (Almeida, Estanqueiro & Salgado, 2016; Belum et al., 2013; Cheung & Chee, 2009; Guerrier & D’Ortenzio, 2013; Kadam et al., 2015; Pound & May, 2005; See, Scott & Levin, 2005), which is essentially an uncontrolled cytokine storm (see Section X) caused by the rapid release of inflammagenic cell wall materials from microbes, often following bactericidal antibiotic treatment (Lepper et al., 2002)."
"The inflammagenic potency of LPS is so great that it is commonly (and ironically) even used as a model to induce symptoms more or less similar to many of the inflammatory diseases of interest. Typically this involves injecting LPS at the site of interest for such diseases. Examples of the use of endotoxin in this way include pre-eclampsia (Cotechini et al., 2014; Faas et al., 1994; Faas et al., 2000; Lin et al., 2012; Liu et al., 2017; Rademacher, Gumaa & Scioscia, 2007; Sakawi et al., 2000; Williamson et al., 2016; Xue et al., 2015), Alzheimer’s (Zhan et al., 2015, 2016), Parkinson’s (Barnum & Tansey, 2010; Byler et al., 2009; Cunningham et al., 2005; He et al., 2013; Hoban et al., 2013; Hritcu & Ciobica, 2013; Hritcu et al., 2011; Liu&Bing, 2011; Miller et al., 2009; Orr, Rowe & Halliday, 2002; Santiago et al., 2010; Tufekci, Genc & Genc, 2011; Z. Zhang et al., 2012), rheumatoid arthritis (Izui, Eisenberg & Dixon, 1979; Nemeth et al., 1985), atherosclerosis (Khedoe et al., 2013), multiple sclerosis (di Penta et al., 2013; Nguyen et al., 2004), Guillain-Barré syndrome (Prendergast &Moran, 2000), sepsis (Lewis, Seymour & Rosengart, 2016; Remick & Ward, 2005), and stroke (Becker et al., 2005; Doll et al., 2015; Shim & Wong, 2016). This far-from-exhaustive list illustrates well the generality of this phenomenon. In cases of stroke, infection is very common, and leads to a worse prognosis; in some cases antibiotics worsen it further (Becker et al., 2016), consistent with the view that the infecting organisms were already present, and that there is an active role of LPS shedding. We note too that some molecules such as P-type inositol phosphate glycans can act as LPS mimics (Robillard et al., 2016). This is especially well established in pre-eclampsia (e.g. Dawonauth et al., 2014; Kenny & Kell, 2018; Robillard et al., 2016; Scioscia et al., 2012, 2011; Williams et al., 2007) but seems to have been little investigated elsewhere."
"Consistent with the above (and see de Punder & Pruimboom, 2015; Kell & Pretorius, 2015a), Table 3 lists a variety of ‘natural’ (i.e. non-experimental) chronic inflammatory diseases for which it has been shown that steady-state endotoxin (LPS) levels are raised and Table 4 presents examples of diseases in which raised levels of lipopolysaccharide binding protein (LBP) have been observed."
"Gram-positive bacteria have a cell wall structure that differs from that of Gram-negatives both in its number of barriers and in the fact that the cell wall component equivalent to LPS is lipoteichoic acid (LTA). LTA is equivalently capable of producing an inflammatory response. In contrast to LPS, which mainly interacts with toll-like receptor 4 (TLR4) (Balasubbramanian et al., 2017; Hoshino et al., 1999; Kell & Pretorius, 2015a; Lien et al., 2000; Poltorak et al., 1998), LTA stimulates target cells mainly by activating toll-like receptor 2 (TLR2) (Ishii & Akira, 2004; Jiménez-Dalmaroni, Gerswhin & Adamopoulos, 2016; Kawai & Akira, 2011; Kumar, Kawai & Akira, 2011; Kumar et al., 2013; Y. Liu et al., 2014; Mukherjee, Karmakar & Babu, 2016; Oliveira-Nascimento, Massari &Wetzler, 2012; Schwandner et al., 1999; Underhill et al., 1999; Zähringer et al., 2008). The glycolipid anchor of LTA plays a central role, analogous to lipid A of LPS (Morath, von Aulock & Hartung, 2005)."
"LTA species have been rather less studied from the point of view of inflammagenesis than have LPS forms, but they clearly reside in the blood and are inflammagens (Barbero-Becerra et al., 2011; Cinar et al., 2013; Hoogerwerf et al., 2009; Levels et al., 2003; Pirillo, Catapano & Norata, 2015). In some respects (see Section X), LTAs may be even more potent than LPS species (Pretorius et al., 2018a)."
"A variety of small-molecule (Donia & Fischbach, 2015) microbial products besides LPS and LTA, such as long- (Schirmer et al., 2016) and short-chain (Thorburn, Macia & Mackay, 2014) fatty acids, can also lead to or modulate the formation of inflammatory cytokines."
"‘Amyloid’, more specifically an amyloid protein fibril, is defined formally (Sipe et al., 2014, p. 221) as ‘a protein that is deposited as insoluble fibrils, mainly in the extracellular spaces of organs and tissues as a result of sequential changes in protein folding that result in a condition known as amyloidosis’. As with prions (Aguzzi & Lakkaraju, 2016; Kell & Pretorius, 2017a; Prusiner, 1998; Prusiner et al., 2015), there is (or need be) no change in the primary sequence when a normally soluble protein adopts an insoluble amyloid form. Anfinsen’s (1973) classical experiments had implied that the primary sequence alone can be sufficient to guide normal folding and that folding was to the state of lowest free energy. The existence of more stable conformations than those first formed upon folding implies, in contrast to this, that there is a large kinetic barrier between the most common conformation and the folded amyloid form(s) of lower free energy (Cohen & Prusiner, 1998) (Fig. 6). As many as 50 ‘amyloid’ diseases are now established (Ankarcrona et al., 2016; Buell, Dobson & Knowles, 2014; Dobson, 2013; Hung et al., 2016; Ke et al., 2017; Kholová & Niessen, 2005; Knowles, Vendruscolo & Dobson, 2014; Siakallis, Tziakouri-Shiakalli & Georgiades, 2014), in which normally soluble proteins fold to form unusual, insoluble amyloid fibril forms and may become on and off-pathway oligomers that are particularly important for cytotoxicity (Ke et al., 2017)."
"Even proteins not normally seen as amyloidogenic or disease-causing can form amyloids; this is of significance in the storage of biological materials, whose shelf-life may be shortened as a result [e.g. insulin (Nielsen et al., 2001,b,c; Wang, 2005)]."
"[..]amyloidogenesis could be induced to occur by the addition of what is stoichiometrically an astonishingly low ratio of bacterial lipopolysaccharide (LPS):fibrinogen, 1:10^8."
"As well as its role in inducing inflammatory cytokine production, there is some evidence that LPS, albeit commonly bound to proteins that can sequester it, is itself directly cytotoxic [reviewed by Kell & Pretorius (2015a) and Williamson et al. (2016)]."
"A general feature of the blood of patients with these chronic inflammatory diseases is that it is both hypercoagulable and hypofibrinolytic (Kell & Pretorius, 2015b); clots form more easily, are stronger, and are less susceptible to proteolysis. The latter is, of course, a particular hallmark of prions (Basu et al., 2007; Saá & Cervenakova, 2015; Saleem et al., 2014; Silva et al., 2015; Woerman et al., 2018) and of amyloids generally (Rambaran & Serpell, 2008)."
"It is hard to disentangle diseases caused or exacerbated directly by inflammation from those where the mediating agent is explicitly a cytokine."
"The different steps considered herein are entirely generic at a broad level (microbes and their dormant states, iron dysregulation, amyloid formation), with differences only apparent at a finer scale (microbial species and the anatomical location of the various dysregulations). The conditions considered herein are all chronic inflammatory diseases, often with quite slow kinetics, and are all in effect diseases of ageing (e.g. van Beek, Kirkwood & Bassingthwaighte, 2016)."
@Captain @YellowFish
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Whenever authors decide to include references within the text, they need to have mercy to facilitate it for the reader (Me, 2019; Myself, 2019; Irene, 2019), making these less interfering with readability.
