Statistical analysis was carried out with Prism R 6.0 (GraphPad Software Inc., La Jolla, CA, USA). Assuming a Gaussian distribution, parametric ANOVAs for independent samples followed by Fisher's post hoc tests were used to compare measurements among the different groups. To evaluate the diagnostic accuracy of the Aβ1−42/Aβ1−40 ratio, a receiver operating characteristic (ROC) curve was generated. Pearson's correlation coefficient (r) was calculated to investigate the associations between different analytes. The results are presented as the mean with standard deviations and were considered to be significant at a p-value < 0.05.

### RESULTS

### Study Population

Samples were collected according to the same standard operating procedures at the Department of Psychiatry, Erlangen, and the Neurology Departments in Erlangen and Munich. Due to the ages at which the different pathologies typically occur, the samples could not be matched for age. However, there was no statistically significant age difference between the patients with meningitis and those with AD or between the controls and the patients with MS. The patients with meningitis had increased peripheral leucocyte counts, increased levels of C-reactive protein (CRP) and CSF protein, cells and erythrocytes and a higher CSF/serum albumin ratio. Patients with MS had slightly increased CSF leucocyte counts and positive oligoclonal bands in the CSF (**Table 1**).

### Reduced Aβ1−42 in AD, MS and Meningitis

All measurements were performed in the same laboratory to avoid inter-center variations. Aβ1−40 and Aβ1−42 levels were quantified by ELISA. One patient in the meningitis group had an Aβ1−42 concentration below the lower limit of quantification and was excluded from further analysis. As expected, a reduced concentration of Aβ1−42 was found in AD samples. Additionally, in MS and bacterial meningitis, concentrations of Aβ1−42 were reduced (**Figure 1A**). The concentration of Aβ1−40 also tended to be reduced in MS and meningitis but not in AD (**Figure 1B**). As a consequence, the ratio of Aβ1−42/Aβ1−40 was reduced only in AD (**Figure 1C**). No significant correlations between the increased TABLE 1 | Patient characteristics—Patient characteristics are presented as the mean (standard deviation).

N, number; CON, control; MS, multiple sclerosis; AD, Alzheimer's disease; CRP, Creactive protein; OCB, oligoclonal IgG bands on isoelectric focusing; CSF, cerebrospinal fluid.

values of erythrocytes, leukocytes or protein and the reduced concentrations of CSF Aβ1−40 or Aβ1−42 were found in patients with meningitis. The established threshold of 0.05 allowed for the differentiation of AD patient samples from control samples and samples from MS and meningitis patients with 100% sensitivity and 91% specificity.

### Reduced Total Amyloid in MS and Meningitis but Not AD

CSF from representative patients with bacterial meningitis, multiple sclerosis and AD as well as one control was loaded directly onto SDS gels containing 8 M urea (**Figure 2A**). To control for artificially low concentrations of Aβ peptides due to the increased protein concentrations in meningitis, an additional separation of CSF from two patients with meningitis and two controls after immunoprecipitation with the 6E10 antibody was carried out (**Figure 2B**). The detection was performed with the anti-amyloid 82E1 antibody, which is specific for Aβ1−x variants.

Reduced concentrations of all Aβ peptide variants were observed in the Western blots of CSF from meningitis patients (**Figure 2**). The concentration even dropped below the lower limit of detection.

### DISCUSSION

The concentration of Aβ1−42 in CSF is reduced in patients with AD, MS and bacterial meningitis. However, the ratio of Aβ1−42/Aβ1−40 is reduced only in AD, while the concentrations of all Aβ peptide variants are similarly reduced in the CSF of patients with bacterial meningitis.

This is the first study to show the distinct changes in Aβ peptide variants in the CSF during AD compared to inflammatory brain diseases.

Nevertheless, the study has several limitations. The sample size is relatively small, and an exact matching of age and sex was not possible. Sample matching is nearly impossible for age because AD is usually diagnosed above the age of 60, while patients with MS become symptomatic before the age of 50. However, the CSF concentrations of Aβ

peptides have been reported to be relatively stable throughout life and to be independent of sex in healthy subjects (Resnick et al., 2015).

To reduce the possible center effects, a balanced composition of the control and the meningitis groups was achieved. However, the samples for the MS group were all collected in Munich, while the samples for the AD group were all collected in Erlangen. However, the preanalytical handling of the samples was identical at all sites, and measurement of the Aβ peptides by ELISA was carried out in a single laboratory, as was recommended to effectively reduce center effects (Mattsson et al., 2013).

While Aβ1−42 is reduced in all three diseases, the observation that the Aβ ratio is reduced in AD but not in meningitis or MS points to a better diagnostic performance of the Aβ ratio. This improved performance has also been previously shown for the differential diagnosis of AD vs. non-neurodegenerative neuropsychiatric diseases, Lewy-body dementia, Parkinson's disease dementia or vascular dementia (Lewczuk et al., 2015b; Janelidze et al., 2016). Several others have therefore suggested to replace the measurement of Aβ1−42 with the Aβ ratio for AD diagnosis (Blennow and Zetterberg, 2015). Our study suggests that the Aβ ratio not only is able to increase the diagnostic performance in the differential diagnosis of neurodegenerative diseases but also helps to differentiate AD from neuroinflammatory diseases.

In the context of the infection hypothesis of AD, the observed differences among AD, MS and meningitis point to different pathophysiological processes behind the changes in CSF Aβ1−42 concentrations. While the processing of APP in AD is changed in a way that leads to an imbalance between the different Aβ peptide variants and allows the accumulation of Aβ1−42 into amyloid plaques, it was hypothesized that APP processing is impeded during CNS infections (Krut et al., 2013). Reduced concentrations ofsecreted (s)APPα and sAPPβ during meningitis support this interpretation (Krut et al., 2013). An alternative explanation is that serum proteins entering the CSF after breakdown of the blood brain barrier in meningitis might mask the detection of Aβ peptides. However, others have found no differences in Aβ peptide concentrations via ELISA after adding proteins to the CSF (Bjerke et al., 2010), and our data show no correlation between increased CSF protein concentrations and reduced concentrations of Aβ peptides. Additionally, we performed immunoprecipitation to release the Aβ peptides from their protein bonds. We suggest that the amyloid peptides build complexes around the invading pathogens and are therefore no longer measurable in the CSF (Spitzer et al., 2016). By agglutinating microorganisms and exerting a direct antimicrobial activity, Aβ peptides have been shown to accumulate around microorganisms in vitro (Soscia et al., 2010; Torrent et al., 2012; Kumar et al., 2016; Spitzer et al., 2016). The increased expression and distinct processing of APP during immunological activation in vitro also favors the idea of an immunological function and an increased consumption of Aβ peptides during infections and neuroinflammation in MS (Maler et al., 2008; Sondag and Combs, 2010; Spitzer et al., 2010). Further research is necessary to analyze the composition and fate of the Aβ peptides accumulating around the invading pathogens.

Current knowledge of inflammation-induced changes in Aβ peptide metabolism is mainly based on studies of acute infections (Spitzer et al., 2010; Kumar et al., 2016). Although MS is not an infectious disease, we used samples from MS patients to investigate the Aβ peptide levels in a chronic inflammatory disease. Mattsson and his colleagues measured the levels of Aβ38, Aβ40, and Aβ42 in patients with chronic neuroborreliosis (Mattsson et al., 2010). In both studies investigating two different entities causing chronic neuroinflammation, the concentrations of all measured Aβ peptide variants were reduced (Mattsson et al., 2010). Therefore, it seems that chronic neuroinfection and neuroinflammation are also associated with reduced levels of all Aβ peptides.

Taken together, the results of this study point to different mechanisms resulting in reduced Aβ1−42 levels in the CSF of patients with AD, bacterial meningitis or MS. The Aβ1−42/Aβ1−40 ratio may help to distinguish AD from neuroinflammatory diseases. Further studies are needed to confirm these findings.

### REFERENCES

### AUTHOR CONTRIBUTIONS

PS, RL, UK, PL, NE, and JM designed the study. PS, JK, TO, PL, UK, HH, and JM investigated the patients and collected the samples. PS, PL, TO, and NE carried out the experiments. The statistics were carried out by PS. PS and JM drafted the manuscript. All authors critically reviewed the manuscript and provided constructive comments to improve the quality of the manuscript. All authors have read and approved the final manuscript.

### ACKNOWLEDGMENTS

This work is supported by grants from the Interdisciplinary Center for Clinical Research (IZKF), Erlangen. The research leading to these results also received support from the Innovative Medicines Initiative Joint Undertaking under EMIF grant agreement no. 115372, resources of which are composed of financial contribution from the European Union's Seventh Framework Program (FP7/2007- 2013) and EFPIA companies' kind contributions. We acknowledge support from Deutsche Forschungsgemeinschaft (DFG) and Friedrich-Alexander-Universität Erlangen-Nürnberg (FAU) within the funding program Open Access Publishing.

**Conflict of Interest Statement:** PL received consultation and lectures honoraria from Innogenetics, IBL International, AJ Roboscreen, and Roche.

The other authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Spitzer, Lang, Oberstein, Lewczuk, Ermann, Huttner, Masouris, Kornhuber, Ködel and Maler. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# It's Never Too Early or Too Late—End the Epidemic of Alzheimer's by Preventing or Reversing Causation From Pre-birth to Death

Clement L. Trempe1 and Thomas J. Lewis 2 \*

1 Independent Clinician, Nahant, MA, United States, 2RealHealth Clinics, Jefferson City, TN, United States

The path to sporadic Alzheimer's is a tragic journey beginning prior to birth and ending in the most dreaded disease of society. Along the disease path are a myriad of clues that portend AD, many of which are complaints of seemingly unrelated conditions from chronic migraines, mood disorders, eye diseases, metabolic syndromes, periodontal diseases, hormonal and autoimmune diseases. Properly treating, not just managing, these diseases, prior to onset of dementia, may significantly reduce dementia incidences. Current high levels of health complaints reflect a state of generalized poor health and compromised immunity. During the mid-Victorian era, people were long-lived yet healthy, suffering from chronic diseases at one tenth the rate of peoples today. It's our poor health, at any age that increases susceptibility to chronic diseases and Alzheimer's. Infection is involved in many cases of Alzheimer's and other neurodegenerative diseases but is also implicated in many chronic conditions. Scientists looking for causation recognize that Alzheimer' is multifactorial and systemic—not "brain only." To slow, stop and reverse the AD epidemic, identification and reversal of causal factors must occur across the entire life spectrum of humans. This approach simply gives consideration to enhancing immune status of our bodies and brain, and controlling inflammation and infection, throughout the entire age spectrum. Infection is a causal factor, but the root cause is multi-factorial and immune health related. Pasteur stated it best when acknowledging the work of Bernard in 19th Century France, "The seed is nothing, the soil is everything."

Accepted: 17 June 2018

#### Citation:

Trempe CL and Lewis TJ (2018) It's Never Too Early or Too Late—End the Epidemic of Alzheimer's by Preventing or Reversing Causation From Pre-birth to Death. Front. Aging Neurosci. 10:205. doi: 10.3389/fnagi.2018.00205 Keywords: Alzheimer's, infection, systemic, diabetes, congenital, immunity, prevention, inflammation

### INTRODUCTION: IMMUNITY AND INFECTIOUS DISEASES

Historically, infectious diseases were the cause of morbidity and mortality. Infectious disease arguably continues to be the major driver of morbidity and mortality however this connection is largely ignored because of the occult nature of many of the causative agents and the cryptic cause and effect between organism and disease (Cochran et al., 2000). Dr. Paul Ewald explains how the concept of evolutionary fitness actually points to infection as being the major cause of disease in modern society (Cochran et al., 2000). Evolutionary fitness means the evolutionary success of an organism relative to competing organism. Genetic traits that may be unfavorable to an organism's

#### Edited by:

Kilmer McCully, Harvard Medical School, United States

#### Reviewed by:

Morgan Newman, University of Adelaide, Australia Maria Markaki, Foundation for Research and Technology Hellas, Greece

> \*Correspondence: Thomas J. Lewis<EMAIL_ADDRESS>
Received: 28 March 2018 Published: 12 July 2018

**128**

Trempe and Lewis Stop Alzheimer's Through Lifetime Prevention

survival or reproduction do not persist in the gene pool for very long. Natural selection weeds them out and any inherited disease or trait that has a serious impact on fitness must fade over time. Therefore, in considering common chronic conditions with severe health consequences, we may presume a non-genetic cause. According to Ewald (2002), ''When diseases have been present in human populations for many generations and still have a substantial negative impact on people's fitness, they are likely to have infectious causes.''

Immune system vitality may be the most important risk factor in any chronic disease including Alzheimer's. The World Health Organization in ''Risk Factors of Communicable Diseases'' (World Health Organization, 2011) states, ''Apart from symbiotic coexistence of human with micro-organisms, disease causing organisms breed in man-made unhygienic conditions of air water and soil. People with low immunity, weak, and living in unhygienic conditions are at greater risk for contracting the infections from surroundings.'' This model of disease fits equally well with Alzheimer's and other chronic diseases but has been limited because source of the infection is less obvious and diagnosis is not frequently enough made or considered.

Chronic inflammation is considered a cause of chronic disease, including Alzheimer's (Rogers, 1995; Holmes et al., 2009; Kawai et al., 2018). As defined by Opie (1929) ''Inflammation may be defined as the process by which cells and serum accumulate about an injurious agent and tend to remove or destroy it.'' Chronic inflammation continues to be blamed for tissue damage but this complex cascade, stimulated by internal and external mediators, results in the release of danger signals that promote immune responses to antigens (Rock and Kono, 2008). Chronic, occult infection is a significant stimulator of chronic inflammation (Beatty et al., 1994). Host-pathogen interactions, defined as the importance of the host's susceptibility for a microbe's virulence, must be considered and this interrelationship is not straightforward. Casadevall and Pirofski (1999) proposed six different classes of host-pathogen interactions that helps explain how the relationship between infection, inflammation, immune health and disease, although apparent, may not always yield statistical certainty.

Rivas et al. (2017) explain that the current research paradigm is reductionist yet biological systems combine their limited elements, creating complex structures and solutions involving infectious diseases. The network theory of aging, and particular, inflamm-aging is defined as, ''a global reduction in the capacity to cope with a variety of stressors and a concomitant progressive increase in proinflammatory status,'' contributes to a more non-reductionist assessment of individualized health by measuring ''many'' rather than one or a few (Franceschi et al., 2000). The construct of inflamm-aging is a measure of immune system activity against chronic insult. Any chronic disease, then, is potentially a measure of the stress on the biological system and its ability, or lack thereof, to cope.

Chronic disease incidences, including Alzheimer's, increases with older age and are linked to immunosenescence

(Solana et al., 2012). Numerous studies show that the pathology of Alzheimer's disease is present decades before a clinical diagnosis of dementia can be made (Mortimer et al., 2005). Predisposition to Alzheimer's, therefore, is established prior to the acceleration of immunosenescence that starts around age 65 (**Figure 1**). The curve also represents the vulnerability to disease due to an immature immune system during the ages 0–5. It is during this time that the antecedents of Alzheimer's and other chronic diseases, specifically occult infections, may opportunistically infiltrate such vulnerable hosts only to express as disease across the spectrum of time and lead to the a significant upswing in Alzheimer's. A comparison of a healthy and unhealthy person is also represented as are transient susceptibilities that may include acute health or sudden life changing events that adversely impact health.

There are numerous occult ectopic obligate intracellular pathogens linked to Alzheimer's, including Chlamydophila pneumoniae (CP; Balin et al., 1998), Borrelia burgdorferi (BB; Marquand and Muller, 1997), gram-negative anerobic bacteria of the oral cavity (Riviere et al., 2002), h-pylori (Malaguarnera et al., 2004), rickettsias (Morrison et al., 1991) and Toxoplasma gondii (Jung et al., 2012). Many infectious species are ubiquitous within both human and animal populations. CP expresses seroprevalence rates of over 50% among adults in the United States (Blasi et al., 2009). In some vulnerable individual, CP migrates to the brain and contributes to neurodegeneration (Gérard et al., 2006).

The connection between immune system health and infection extends back to the research of Bernard and Pasteur (Garko, 2012). Modern studies at MIT demonstrate a strong correlation between immunity and future Alzheimer's (Dougherty, 2015). The CDC, in an article on neglected parasitic infections explain how, of the 60 million Americans infected with the Toxoplasma parasite, only a small proportion of the susceptible experience severe health consequences (Wong and Remington, 1994; Chaudhry et al., 2014; Jones et al., 2014). Higher prevalence of T. gondii in patients with AD compared to controls shows an association between organism and disease (Schulz et al., 2007; Rashno et al., 2016, 2017). An estimated 18% of the United States are seropositive and worldwide, an estimated one third of the population are infected (Dalimi and Abdoli, 2012). Chronic infection is reported to reactivate toxoplasmosis (Montoya and Liesenfeld, 2004). For the individuals infected, any susceptibility factor illustrated in **Figure 1** may contribute to toxoplasmosis proliferation and potential exacerbation of Alzheimer's.

The CDC indicates that few infected with toxoplasmosis have symptoms. Yet, according to Kaiser Family Foundation, the highest percentage of medical spending occurs for ill-defined conditions (Peterson-Kaiser Health System Tracker, 2018). Toxoplasmosis and other occult organism likely contribute to these conditions and associated high cost of care. Stratton and Wheldon (2006) explains the life cycle of CP and its treatment when discovered associated with multiple sclerosis and heart diseases. Dr. Wheldon contributed the following list of disease presentations he associated with CP in his clinical practice over 20 years: cardiac conduction defects; effusive pericarditis with tamponade; chronic obstructive airways disease; multiple sclerosis; Alzheimer's disease; chronic fatigue syndrome; encephalitis; retinal vasculitis; macular degeneration; progressive presbyopia; Crohn's disease; new onset adult asthma; and schizophrenia (hebephrenia). A similar list associated with Toxoplasmosis may be established with more research.

Lyme disease, caused by vector spirochetes, is a wellrecognized chronic disease precipitated from BB and co-infections. Miklossy hypothesized on a link between BB and Alzheimer's in 1994 and proved a causal relationship (Miklossy, 1994, 2011). The CDC recently revised its estimate of 30,000 new cases per year in 2014 to 300,000+ in 2017 in the United States Kris Kristofferson was diagnosed with AD yet, after a discovery of Lyme disease, the public message was, ''Kristofferson struggled with memory problems in recent years and was told he had Alzheimer's disease, but it appears he was misdiagnosed and all along has actually been suffering from the tick-borne illness Lyme disease'' (Marcus, 2016). The association and causation between infection and Alzheimer's continues largely disregarded.

In ''Overview of Lyme Disease: A Critique of an Ignored Pandemic,'' Dr. Ken Stoller suggests that 20% of the human population is infected and may well explain diseases of unknown origin (Stoller, 2015). In a recently completed investigation of a cohort of 70 functioning but unhealthy manufacturing company workers in central Indiana, we evaluated 41 for stealth infection, including Lyme disease, based on reported lifestyle, symptoms, a broad array of inflammatory biomarkers, and serology testing. Of the 41, 40 were positive for at least one occult organism and most had two or more, 28 were found to have positive IGG titers for CP; 12 were positive for Toxoplasma Gondii; 18 were positive for at least one IGG band for Lyme; one was positive for Q Fever and one was positive for Rickettsia Typhii (Lewis et al., submitted). Presuming that the 30 untested individuals were negative for Lyme, these data support the estimated population infectious burden suggested by Stoller.

## PRE-BIRTH SUSCEPTIBILITY TO FUTURE AD

Congenital infections affect the unborn fetus or newborn infant. Conventionally, they are thought to be caused by viruses that may be picked up by the baby at any time during the pregnancy. Even if the mother is known to have a viral illness during her pregnancy, her immune system may prevent the virus from infecting the fetus or newborn infant. However, shortly after birth for mothers that do not breast feed or shortly after stopping breast feeding, the newborn's immune system function drops precipitously and then slowly strengthens from ages 0–5, **Figure 1**.

Periodontal bacteria is a significant contributor to congenital infection. Offenbacher et al. (1996) were the first to report a relationship between maternal periodontal disease and delivery of a preterm infant. Global incidence of preterm birth is around 9.6% with regional disparities from 12% to 13% in USA, 5% to 9% in Europe, and 18% in Africa. According to ''Born Too Soon,'' the United States ranked 131st out of 184 in preterm births (Blencowe et al., 2013). Full term stillbirth is also caused by bacteria including Fusobacterium nucleatum (Han et al., 2010). The placenta is an immuno-suppressed organ compared to other organs like the liver and the spleen which makes it easy for the bacteria to colonize there. Intrauterine infection is considered a leading cause of early preterm birth and data from clinical and experimental studies suggest that infection accounts for upward of 40% of these high-risk preterm deliveries (Kravetz and Federman, 2005; Kemp et al., 2017).

Determining a statistical association between periodontal infection induced premature births and dementias is challenging because of the 70+ year gap between birth and disease. In ''Birth in a High Infant Mortality State: Race and Risk of Dementia,'' higher rates of dementia later in life was statistically connected to higher incidences of infant mortality in racial cohorts (Gilsanz et al., 2017). In a cohort of elderly individuals, blacks born in states with the highest levels of race specific infant mortality rates had 40% increased risk of dementia compared to national averages. Early life conditions may contribute to racial inequalities in dementia incidence according to the authors. Since infection accounts for a preponderance of preterm deliveries, one can surmise that the excess of dementia is tied back to either a failure to thrive at birth (poor immune status), chronic infection acquired during pregnancy, or a combination of these adverse circumstances, all of which add risk for future Alzheimer's upon immunosenescence (Jeffcoat et al., 2002), **Figure 1**.

Non-periodontal infections are connected with both adverse pregnancy outcomes and dementias. Cytomegalovirus (CMV) infection in pregnancy causes adverse clinical outcomes and the rate of transmission in utero was reported at roughly 33% (Stagno et al., 1987). CMV is present in a very high proportion of brains from vascular dementia patients (Lin et al., 2002). It has also been implicated as a risk factor for Alzheimer's (Barnes et al., 2014). Besides the inflammatory response elicited by infection, CMV may also drive immunosenescence and thus make an individual more susceptible to Alzheimer's as a result of the infection or a co-infection acquired during any time in lifespan, **Figure 1**. According to Dow, ''In immune competent individuals, infection with CMV is usually asymptomatic, even in neonates, but once established, its containment becomes a priority for the immune system, which is unable to completely eliminate it (Dow, 2015). However, even healthy immune competent people may display symptoms of CMV infection more often than previously appreciated, sometimes even with serious consequences and with age implicated as a risk factor. The consequences to the immune system for maintaining this constant CMV vigilance may be severe. Reports on the very young and the very old show that CMV infection results in similar alterations to CD8+ T cell subset surface phenotypes. This has given rise to the concept that what are apparent age-associated changes could rather be due to age-associated increases in prevalence of CMV infection.'' This phenomenon may be driven by other pathogens as well with susceptibility and disease incident being explained by the proposed different classes of host-pathogen interactions (Casadevall and Pirofski, 1999).

### EARLY LIFE SIGNS AND SUSCEPTIBILITY TO FUTURE AD

In the United States, the peak age at diagnosis for Type 1 diabetes (T1D) is 10–14 (Maahs et al., 2010). Genetics may play a role, but infection is strongly associated with the condition. T1D follows viral infections such as mumps, rubella, CMV, measles, influenza, encephalitis, polio, or Epstein-Barr virus. Viruses play an important role in the pathogenesis of T1D by inducing or accelerating the beta cell destruction process (Christen et al., 2012). Bacteria are also T1D associated agents. CP was found in 46.5% of young patients with T1D compared to 10.5% of non-diabetic controls (Rizzo et al., 2012). Additionally, CP antibody positivity was significantly more common in patients in poor metabolic control (HbA1c >9%) vs. patients in good metabolic control (HbA1c <7%) and dysfunction of pancreatic beta cells (Rodriguez et al., 2015). Older patients with T1D have more than an 80% increased risk for dementia compared with those without diabetes and the risk is even high when T2D subjects are excluded (Whitmer et al., 2015). Thus T1D, and more importantly the potential immune dysfunction and infection associated with the disease, are indicators of future AD.

### YOUNG LIFE SIGNS AND SUSCEPTIBILITY TO FUTURE AD

More than 10% of all primary care office visits are depressionor mood-related (Stafford et al., 2000; Olfson et al., 2014). The research community knows that depression and Alzheimer's disease are linked and its now clear that depression is a risk factor, not just a symptom, of AD (Dantzer et al., 2008; Wilson et al., 2010) A 17-year prospective study from the Framingham cohort demonstrates that older adults with depressive symptoms are at a 50% increased risk of developing Alzheimer's disease (Saczynski et al., 2010). In another study, the authors showed an incident AD group compared to those without AD reported a barely perceptible increase in depressive symptoms during 6–7 years of observation before the diagnosis and no change during 2–3 years of observation after the diagnosis (Wilson et al., 2010). According to Caraci et al. (2010) the molecular mechanisms and cascades that underlie the pathogenesis of major depression, such as chronic inflammation and hyperactivation of the hypothalamic-pituitary-adrenal (HPA) axis, are also involved in the pathogenesis of AD (Caraci et al., 2010). Johnson et al. (2015) found that the signs of depression begin well before the symptoms of dementia begin manifesting. They believe that there are a core group of mood and depression symptoms, easily measureable in populations, that may enable medical professionals to differentiate between people at risk of developing dementia and normally aging individuals.

In ''Latent Toxoplasmosis and Humans,'' the authors review the impact of toxoplasmosis in the etiology of different mental disorders including schizophrenia and depressive disorders, obsessive-compulsive disorder, Alzheimer's and Parkinson's disease, epilepsy, headache and migraine, mental retardation, suicidal tendencies and intelligence (Dalimi and Abdoli, 2012). Toxoplasma gondii impairs memory in infected seniors without diagnosis of neurodegenerative disease. In a study of executive function and memory, subjects positive for Toxo showed memory performance reduction of about 35% compared to the toxo negative group and this group also reported a decreased quality of life compared to those not infected (Gajewski et al., 2014). In our own study cohort of 70 individuals, 40 were tested for IgG antibodies for toxoplasmosis and 12 were positive (Lewis et al., submitted).

The causal agent of Lyme is reported in mood disorder of young and middle-aged people (Fallon et al., 1993; Dersch et al., 2015). Determining causation from infection in mood disorders and suicidal tendencies suffers from crypticity. Garakani and Mitton (2015) state, ''The patient's panic attacks resolved after he was discharged and then, months later, treated with long-term antibiotics for suspected ''chronic Lyme Disease'' despite having negative Lyme titers'' (Garakani and Mitton, 2015). A significant feature of post-treatment Lyme disease syndrome is depression (Rebman et al., 2017). Miklossy has provided adequate analysis to convince the most stubborn opponent of the relationship between BB and Alzheimer's (Miklossy, 1994, 2011; Miklossy et al., 2004). Depression may be a clue as to the same infectious process and since depression generally precedes Alzheimer's, treatments may be more effective.

### YOUNG TO MIDDLE LIFE SIGNS AND SUSCEPTIBILITY TO FUTURE AD

The Rotterdam study revealed an increased risk of dementia and AD in patients with type 2 diabetes (T2D; Ott et al., 1999). Those with T2D are at 50% greater risk of developing a neurodegenerative condition compared to healthy contemporaries (Mayeda et al., 2015). Insulin resistance early in life may enhance the risk of developing neurodegeneration (Luciano et al., 2015). Insulin resistance is due in part to infection, as first described in 1943 (Greene and Keohen, 1943). The article published in JAMA titled, ''Insulin resistance due to infection in diabetes mellitus in man,'' has not been widely cited. A modern version concludes that pathogen burden has the strongest association with insulin resistance among all the risk factors considered (Fernández-Real et al., 2006). For example, HSV-2 titer was negatively associated with insulin sensitivity even after factoring for inflammation. The relationship was strengthened further for subjects that were seropositive for CP and Enteroviruses. People with T2D are more prone to be on the ''high susceptibility individual'' curve of **Figure 1**.

 Thus, anatomically and developmentally, the retina is an extension of the CNS; it consists of retinal ganglion cells, the axons of which form the optic nerve, whose fibers are, in effect, CNS axons. The eye has unique physical structures and a local array of surface molecules and cytokines, and is host to specialized immune responses similar to those in the brain and spinal cord. Several well-defined neurodegenerative conditions that affect the brain have manifestations in the eye, and ocular symptoms often precede CNS diagnoses by up to 20 years (London et al., 2013). Various eye-specific pathologies share characteristics of other CNS pathologies, including Alzheimer's disease and glaucoma (McKinnon, 2003).

Immunoglobulin serology for CP is significantly higher in patients with glaucoma compared to controls (Yuki et al., 2010). Other infections noted in association with glaucoma include: Helicobacter pylori (Galloway et al., 2003), Bartonella henselae (Gray et al., 2004), HIV (Nash and Lindquist, 1992), CMV (Sekhsaria et al., 1992), Toxoplasmosis (Sheets et al., 2009), Tuberculosis (Egbagbe and Omoti, 2008), and Lyme disease (Zaidman, 1997). The eye is susceptible to inflammatory diseases through a delicate balance with survival coined ''ocular immune privilege.'' According to Streilein, ''Not surprisingly, inflammation, if it occurs within the eye, is a profound threat to vision'' (Hazlett and Stein-Streilein, 2012). Immune privilege and easy accessibility provides an explanation why neurodegeneration presents in the eye long before the brain in many cases and offers a low-cost early or pre-disease screening tool and link to treatable associations to future AD.

Macular degeneration is both a vascular and neurodegenerative disorder easily observed with simple yet elegant optometric instruments. Studies titled, ''Age-related macular degeneration (AMD): Alzheimer's disease in the eye?'' (Kaarniranta et al., 2011) and ''Parallel findings in age-related macular degeneration and Alzheimer's disease,'' (Ohno-Matsui, 2011) provide examples of what early neurodegenerative disease diagnosis could be. The less studied common denominator between macular disease and neurodegenerative diseases is infection. CP is a documented actor in age-related macular degeneration (Haas et al., 2009). Our own clinical evaluations, being compiled for publication, show seropositive CP and other occult infections in significant percentages of clinical AMD cases.

### INFECTION, INFLAMMATION AND ALZHEIMER'S "HALLMARKS"

Balin et al. (1998) links CP to Alzheimer's. Subsequently they have published dozens of articles on this topic including, ''Proof of Concept Studies of Chlamydia Pneumoniae Infection as a Trigger for Late-onset Alzheimer Disease'' (Balin, 2017). A Harvard-led team was the first to definitely show that the beta amyloid hallmark of AD is actually has antimicrobial properties (Soscia et al., 2010). Ravnskov and McCully explained that microvascular hypoxia is initiated by the sequestration of infectious remnants by LDL particles and this process is also noted in Alzheimer's (Bailey et al., 2004; Ravnskov and McCully, 2009). Tau hyperphosphorylation is also noted in microvascular disease-associated neurodegenerative diseases (Castillo-Carranza et al., 2017). Hibernating animals also hyperphosphorylate tau and the common denominator between Alzheimer's and hibernation may be hypoxia and, like with beta amyloid, the modified tau serves to protect the brain (Su et al., 2008). Corriveau et al. (2016) explain the significance of vascular contributions to cognitive impairment and dementia. Miklossy et al. (2006) noted increase in inflammation, beta-amyloid precursor protein, and hyperphosphorylated tau production induced by spirochetes or lipopolysaccharide. Balin (2017) delved into the link between the pathogens, inflammation, Abeta 1–42 production, regulation of gene transcripts, and protein expression. The conclusions regarding gene transcripts and protein expression are supported by studies of infectogenomics (Kellam and Weiss, 2006; Nibali et al., 2014). No other single explanation, other than infection, supports all the observed pathologies in Alzheimer's.

### CONCLUSION

Sub-optimal health and chronic inflammation predisposes individuals to opportunistic infections and a myriad of diseases throughout the continuum of life provide information about risk and susceptibility to future Alzheimer's disease. Poor health status, periodontal diseases, congenital infections, depression, diabetes, eye diseases, to name a few, provide a roadmap of progression towards neurodegenerative disorders. Fundamental to the onset and propagation of all these diseases is immune system immaturity, dysfunction and senescence that facilitate proliferation of opportunistic infection. Clinically, medicine must change its paradigm of managing disease symptoms and adopt a proactive approach of testing for immune health status, by way of biomarkers of inflammation, to identify those at risk of future Alzheimer's and other chronic diseases. Next, each at-risk subject must be provided guidance on how to improve their health status. In addition, those with compromised immune status must also be tested for infection, and if found, treated until the burden is lowered or eradicated. This program must be conducted across the age spectrum for people showing signs and symptoms of any disease associated with Alzheimer's in later life. This approach could conceivably delay Alzheimer's onset by 5 years which could reduce number of incidences in half. When clinicians recognize association and causation between earlier chronic conditions and Alzheimer's and focus on mitigation rather than management, a 50% or greater reduction in the disease becomes realistic.

### REFERENCES

### AUTHOR CONTRIBUTIONS

TL and CT co-authored this work. The inspiration for this article is the clinical work of CT. TL is responsible for the research and assembly of the document.

### FUNDING

This work was funded exclusively by the authors with no outside support.

transmission to fetus, and clinical outcome. Obstet. Anesth. Digest. 7:46. doi: 10.1097/00132582-198707000-00007

**Conflict of Interest Statement**: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Trempe and Lewis. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Can an Infection Hypothesis Explain the Beta Amyloid Hypothesis of Alzheimer's Disease?

Tamas Fulop1 \*, Jacek M. Witkowski2 , Karine Bourgade1 , Abdelouahed Khalil1 , Echarki Zerif1 , Anis Larbi3 , Katsuiku Hirokawa4 , Graham Pawelec5,6, Christian Bocti1 , Guy Lacombe1 , Gilles Dupuis7 and Eric H. Frost8

1 Division of Geriatrics, Department of Medicine, Research Center on Aging, University of Sherbrooke, Sherbrooke, QC, Canada, 2 Department of Pathophysiology, Medical University of Gdansk, Gda ´ nsk, Poland, ´ 3 Singapore Immunology Network, A∗STAR, Biopolis, Singapore, Singapore, 4 Department of Pathology, Nitobe Memorial Nakano General Hospital, Tokyo, Japan, 5 Department of Internal Medicine II, Center for Medical Research, University of Tübingen, Tübingen, Germany, 6 Health Sciences North Research Institute, Greater Sudbury, ON, Canada, 7 Department of Biochemistry, Graduate Programme of Immunology, University of Sherbrooke, Sherbrooke, QC, Canada, 8 Department of Microbiology and Infectious Diseases, University of Sherbrooke, Sherbrooke, QC, Canada

Alzheimer's disease (AD) is the most frequent type of dementia. The pathological hallmarks of the disease are extracellular senile plaques composed of beta-amyloid peptide (Aβ) and intracellular neurofibrillary tangles composed of pTau. These findings led to the "beta-amyloid hypothesis" that proposes that Aβ is the major cause of AD. Clinical trials targeting Aβ in the brain have mostly failed, whether they attempted to decrease Aβ production by BACE inhibitors or by antibodies. These failures suggest a need to find new hypotheses to explain AD pathogenesis and generate new targets for intervention to prevent and treat the disease. Many years ago, the "infection hypothesis" was proposed, but received little attention. However, the recent discovery that Aβ is an antimicrobial peptide (AMP) acting against bacteria, fungi, and viruses gives increased credence to an infection hypothesis in the etiology of AD. We and others have shown that microbial infection increases the synthesis of this AMP. Here, we propose that the production of Aβ as an AMP will be beneficial on first microbial challenge but will become progressively detrimental as the infection becomes chronic and reactivates from time to time. Furthermore, we propose that host measures to remove excess Aβ decrease over time due to microglial senescence and microbial biofilm formation. We propose that this biofilm aggregates with Aβ to form the plaques in the brain of AD patients. In this review, we will develop this connection between Infection – Aβ – AD and discuss future possible treatments based on this paradigm.

Keywords: peripheral innate immune system, Alzheimer's disease, infections, amyloid beta, blood–brain barrier, monocytes/macrophages, biofilms, senile plaques

## INTRODUCTION

Alzheimer's disease (AD) was identified over 100 years ago when Alois Alzheimer and others described the pathological hallmarks of this devastating disease (Alzheimer, 1907; Alzheimer et al., 1995). The histopathological characteristics of AD are extracellular deposition of Aβ and intracellular accumulation of the tau protein in a hyperphosphorylated form (Hanger et al., 2014;

#### Edited by:

Donna M. Wilcock, University of Kentucky, United States

#### Reviewed by:

Xuemin Xu, University of Texas of the Permian Basin, United States Morgan Newman, University of Adelaide, Australia

> \*Correspondence: Tamas Fulop<EMAIL_ADDRESS>
Received: 31 March 2018 Accepted: 02 July 2018 Published: 24 July 2018

#### Citation:

Fulop T, Witkowski JM, Bourgade K, Khalil A, Zerif E, Larbi A, Hirokawa K, Pawelec G, Bocti C, Lacombe G, Dupuis G and Frost EH (2018) Can an Infection Hypothesis Explain the Beta Amyloid Hypothesis of Alzheimer's Disease? Front. Aging Neurosci. 10:224. doi: 10.3389/fnagi.2018.00224

Sun et al., 2015) leading to synaptic dysfunction which highly correlates with the cognitive decline (Terry et al., 1991). These findings gave rise to what is called the "amyloid cascade hypothesis of Alzheimer's disease" (Beyreuther and Masters, 1991; Hardy and Allsop, 1991; Karran and De Strooper, 2016). Neuroinflammation is regularly observed in AD and has been incorporated into the amyloid hypothesis (Rogers et al., 1992; McGeer and McGeer, 2013; Bolós et al., 2017). However, despite the discovery of these pathological hallmarks almost 100 years ago and the belief that they are also at the origin of the disease, no progress has been made in AD treatment (Mehta et al., 2017; Sacks et al., 2017). Thus, a new paradigm is needed to integrate the knowledge accumulated through the decades and which can lead to further discoveries and effective treatments that are so urgently required.

### WHAT IS ALZHEIMER'S DISEASE?

### Clinical Aspects

Alzheimer's disease is one of the most deleterious neurodegenerative diseases known (Tam and Pasternak, 2012). Clinically, AD can be well-defined and distinguished from other dementias. Starting with memory problems, it progresses inexorably toward the total loss of the patient's identity (Castellani et al., 2010). In our aging society, it has become one of the most disastrous plagues of modern humanity. Whereas the incidence of heart disease and cancer are declining or stable, AD and dementia are expected to almost triple in the next 30 years. However, it is still questionable whether there are as many clinically "pure" forms of AD, presenting only with the typical pathophysiological alterations in the brain cortex and leading to neurodegeneration, as is claimed (Fulop et al., 2013a,b). We still do not know whether most cases diagnosed as AD are in reality a mixed form involving cortical as well as subcortical cognitive changes (Schreiter Gasser et al., 2008), although the recent inclusion of many cardiovascular risk factors seems to support this contention (Gottesman et al., 2017; Falsetti et al., 2018). From the clinical point of view, efforts have been made to redefine AD to accommodate the amyloid hypothesis. Recently, it has been claimed that the initial stages of AD designated as prodromal or pre-clinical stages, or subjective memory complaint (SMC) which still cannot be clinically diagnosed with any certainty, may nevertheless only be detected by changes in the "biomarkers" of AD such as Aβ and pTau levels in the cerebrospinal fluid (McKhann et al., 2011; Reiman et al., 2011; Blennow and Zetterberg, 2018; Ghidoni et al., 2018).

This new and very appealing clinical classification of AD prior to the clinical manifestation of the disease, nevertheless has helped to advance the field, but not exactly as it was intended (Bondi et al., 2017). It has rather stimulated the AD community by making it aware of the fact that AD is not exclusively a disease of the elderly but starts decades before the first clinical symptoms such as mild cognitive impairment (MCI) appear (Le Page et al., 2017). Despite this new classification, all stages of the disease, preclinical and clinical, were linked to the prevailing paradigm of the amyloid hypothesis which states that pathological Aβ production induces a neuroinflammatory state which, once crossing a certain threshold, results in overt AD. This new classification has incorporated the notion of neuroinflammation, recognized many years ago, but again linked to Aβ production and deposition, despite the emerging evidence that neuroinflammation actually precedes Aβ deposition as will be discussed later in this review (McManus and Heneka, 2017).

### Pathological Aspects

Alzheimer's disease was initially described by Alois Alzheimer in a 56-years-old woman who had most probably a familial (hereditary) form of early onset AD according to our present understanding (Alzheimer, 1907; Alzheimer et al., 1995). This form comprises only about 5% of all AD cases, whereas the vast majority are so-called late onset AD, which is more frequent with increasing age (Bekris et al., 2010). Thus, age is considered as one of the most important recognized risk factors for late onset AD (Castellani et al., 2010). From the original observations of Alois Alzheimer on the pathological findings in the cortex of his patient there was a paradigm change in the 80s due to the application of these same pathological findings also as hallmarks of the sporadic form of the disease. Since then, continuing confusion concerning the aetiologies of these two separate pathologies has persisted (Fulop et al., 2013a,b; Bondi et al., 2017). Because the patient described by Alzheimer had amyloid plaques and neurofibrillary tangles, then according to this definition, all AD patients should have the same pathology. Logically this should also be the cause of the disease.

Considerable research efforts have been devoted to the amyloid hypothesis. Initially these efforts were directed toward to identifying what the exact composition of amyloid is and secondly to understand how it is produced. It was established that it originates from amyloid precursor protein (APP) either extracellularly or intracellularly. The normal processing of APP is not amyloidogenic (i.e., does not result in the production of Aβ) and most probably has a physiologic role in synaptic maintenance (Perneczky et al., 2013). However, for some reason(s) which have not been fully investigated, there is a shift in APP processing to an amyloidogenic metabolism and the formation of extracellular Aβ fibrils. These fibrils are the basis of the amyloid plaques already described by Alzheimer. We know that changes in the cellular membranes, and especially the enriched cholesterol contents of their lipid rafts, favors the amyloidogenic pathway (Burns et al., 2006). However, other causes responsible for this amyloidogenic production are known, such as infections but these explanations have not yet gained acceptance in the AD research community (Bourgade et al., 2016a,b).

The most important by-products of amyloidogenic APP metabolism are the beta amyloid peptides 1–42 and 1– 40. The most abundant is Aβ1–40, but the most toxic is Aβ1–42 (Galante et al., 2012). These products can be measured in different compartments of the body in addition to the immunopathological determination in the brain. In cerebrospinal fluid and in the blood, it is claimed that Aβ decreases in clinically diagnosed AD. Interestingly, in blood, Aβ was found to be more abundant during the MCI stage of the disease than in clinically diagnosed AD (Camponova et al., 2017).

Aβ in blood is also claimed to be a biomarker for the early and very early stages of the disease. Many clinical trials were based on its measurement and the hope that treatment to reduce Aβ levels would consequently prevent the progression to full blown AD (Cummings et al., 2018; Molinuevo et al., 2018). Unfortunately, the many clinical trials that aimed at decreasing Aβ levels in the brain by active or passive vaccination including use of monoclonal antibodies did not result in clinically useful results.

The next issue was to investigate what is the precise mechanism of Aβ production. Huge efforts were devoted to this research as according to the amyloid hypothesis, if we decrease its production even if not completely preventing it, the clinical effects should be notable. The normal mechanism was elucidated and was shown to be driven by α-secretase which produced soluble APP fragments (Chow et al., 2010). The enzymes involved in amyloidogenic Aβ production are β-secretase (BACE) and then γ-secretase acting sequentially (Rivest, 2009; Siegel et al., 2017). Many different compounds that inhibit these secretases were tested in phase II and III clinical trails, but without any clinical success. These failures cast further doubts about continuing to adhere to the amyloid hypothesis.

### The Amyloid Hypothesis

This hypothesis identifying amyloid as the possible cause of AD remains the most popular and widely accepted theory, despite its drawbacks discussed above. This hypothesis states that due to a change in proteostasis, as one of the hallmarks of aging (Witkowski et al., 2017), APP is broken down to create Aβ and when this occurs unopposed, then pathology results.  However, it is clear that such senile plaques are commonly present in the brain in the absence of any cognitive pathology. So, how does Aβ act differently in patients compared to healthy people? There are several receptors for Aβ on different cells but mainly on microglia, which are brain macrophages that can engulf Aβ and destroy it. However, with time this process is circumvented, either because Aβ production becomes overwhelmingly increased or because of the "senescence" of the microglia that lose functionality, resulting in Aβ starting to slowly accumulate and deposit in plaques. In parallel, as the microglia are unable to ingest all the Aβ, pathogen or pattern recognition receptors (PRRs) such as the TLRs, CD36, RAGE sense the presence of Aβ and induce a strong inflammatory reaction leading to free radical and pro-inflammatory cytokine production (Su et al., 2016; Venegas and Heneka, 2017; Le Page et al., 2018). This will lead to the well-described neuroinflammation and the destruction of neurons. At the meantime, the Tau protein which is necessary for the maintenance of axon physiology, becomes hyper-phosphorylated because of this inflammatory process, and forms neurofibrillary tangles, which mauls the structure of neuronal processes leading first to degradation of synapses and consequently to neuron death (Serrano-Pozo et al., 2011; Holtzman et al., 2016; Leyns and Holtzman, 2017). Thus, according to this model, Aβ is at the center stage of AD at all disease stages. However, in the absence of any noticeable success of treatments based on the amyloid hypothesis, competing hypotheses urgently require consideration.

### Vascular Hypothesis

The "vascular hypothesis" has been present since the very early 1990s. It states that the production of Aβ may be a consequence of ischemia occurring in the brain with age. Cerebral amyloid angiopathy (CAA) is a major pathological feature of AD where amyloid spreads and deposits throughout the blood vessel walls in the central nervous system. These pathogenic events induce a specific clinical presentation profile including cerebral hemorrhage, stroke, ischemic infarctions, subarachnoid hemorrhage, seizures, cognitive impairment, and dementia (Bu et al., 2013). Epidemiological studies have shown that several well-established risk factors for AD, including diabetes mellitus, atherosclerosis, stroke, hypertension, transient ischemic attacks, microvessel pathology and smoking, have a vascular component that reduces cerebral perfusion (de la Torre, 2002). In fact, detection of regional cerebral hypoperfusion through neuroimaging techniques can preclinically identify individuals at risk for AD. Further, cerebral hypoperfusion precedes hypometabolism, cognitive decline, and neurodegeneration in AD (de la Torre, 2002). Therefore, disturbance of the cerebrovascular system is likely to be a major contributor to AD pathogenesis. This seems very attractive, but there are many vascular lesions in the brain which do not lead to Aβ production and deposition.

Alzheimer's disease might originate from either or both of the amyloid and vascular hypotheses together. Indeed, there could be an intertwined form of AD which is a mixed manifestation where the two pathologies co-exist. Initially one may predominate, but at the end they might become indistinguishable. So, while we can recognize that vascular changes may somehow contribute, these are far from explaining all of the pathogenesis of AD.

### Infection Hypothesis

The infection hypothesis was presented as a hypothetical causative explanation even by Alois Alzheimer himself. This hypothesis was discarded but has more recently been "rediscovered" (Itzhaki et al., 2016). What is the evidence that AD may be of infectious origin?

The resurgence of the hypothesis that microorganisms might have an important role in the development of AD was rekindled by the pioneering work of Itzhaki's group who showed that plaques contain remnants of HSV-1 viral DNA (Wozniak et al., 2007, 2009, 2011; Itzhaki, 2016). This was one of the first attempts to link AD pathological hallmarks to something other than Aβ. The hypothesis proposes that in people infected by HSV-1 (the majority of elderly persons), some show a decline of the immune system with age which enables HSV-1 to migrate from the periphery to the brain, or alternatively,

in stressful circumstances, HSV-1 infects the brain directly via the olfactory route. In the latter case, this infection is mild. Once HSV-1 is in the brain, it is able to facilitate several processes which contribute to neuroinflammation (e.g., direct stimulation of TLRs), as well as to direct neuronal cytopathology and ultimately to neural degeneration (e.g., senile plaque formation). There are experimental data supporting the proposal that HSV-1 in the brain directly contributes to the abnormal processing of APP to Aβ and favors their toxic aggregation and also the hyperphosphorylation of Tau (Itzhaki, 2016). Further experimental data suggest that other viruses, such as CMV, may also be involved in the pathogenesis of AD (Lövheim et al., 2018). More recently the presence of antiviral antibodies in epidemiological studies, especially high IgM levels (representing the reactivation of viral infections), has been correlated with the long-term development of AD (Lövheim et al., 2015). Interestingly, these studies imply that the greater the number of different microorganisms detected in the periphery, the greater the probability of developing AD (Lövheim et al., 2018). This suggests that not one single agent, but a community of microorganisms may be involved in triggering AD (Carter, 2017). These experimental data indicate that such agents may act as environmental pathogenic trigger factors interacting with genetic (e.g., APOE4) and immunologic factors (Costa et al., 2017) to explain the heterogeneity of susceptibility to AD.

A second pioneering group of investigators, invoking the infection hypothesis, has suggested a role for spirochetes in the pathogenesis of AD (Miklossy, 2011a,b, 2016; Miklossy and McGeer, 2016). They demonstrated the presence of Borrelia burgdorferi in the post-mortem brains of many AD patients and showed that senile plaques are biofilms built by these organisms to protect themselves from host defense mechanisms and to assure their own survival (Miklossy, 2016). These observations provided a new impetus to the infection hypothesis as we will describe below. However, the exact composition and the origin of the amyloids contained in plaques/biofilms has not been precisely determined.

Almost at the same time, Balin et al. (1998, 2008) also made the important observation that Chlamydophila pneumonia, an obligate intracellular, Gram negative bacterium was present in post-mortem AD brains. Systemic infection by this pathogen was associated with a fivefold increase in AD occurrence and many AD patients have increased anti-C. pneumonia antibody titers in blood. C. pneumonia may also enter directly through the olfactory tract, as was described for HSV-1, and infect or colonize different cells of the brain including microglia (Bu et al., 2015). Viable bacteria were detected by one study near the plaques in AD brains (Gérard et al., 2006). Dormant reservoirs of bacteria have increasingly been discovered in the body. It was recently demonstrated albeit not in AD that Staphylococcus aureus may survive in some Kupffer cells and as such constitute a dormant reservoir, the reactivation of which may occur at any time when the circumstances become favorable (Surewaard et al., 2016). These infections were related to the ApoE4 genotype, a known genetic risk factor for AD. Since these seminal observations, other bacteria were demonstrated in AD brain such as Pseudomonas aeruginosa, Escherichia coli, Helicobacter pylori, and S. aureus (Singhrao et al., 2015; Zhan et al., 2016). Of note, great care was taken by these investigators to exclude the possibility that the presence of these microorganisms in the brain was due to post-mortem contamination.

Consistent with these data, it is well-recognized that periodontitis and gingivitis are linked to a higher risk of AD (Singhrao et al., 2015; Pritchard et al., 2017). As these are chronic inflammatory diseases affecting the whole body it is not surprising that they are also associated with cardiovascular disorders, type 2 diabetes mellitus and rheumatoid arthritis (Carter et al., 2017; Kriebel et al., 2018) which, in turn are additional risk factors for the development of AD. What is not known exactly is the pathomechanism of this connection and whether specific pathogens are involved in this association. There are many candidates but none of them have been proven. In this context the role of Porphyromonas gingivalis as the "master bacterium" orchestrating the whole community of microorganisms inside the mouth has been strongly evoked (Hajishengallis et al., 2012). This bacterium is able to subvert the role of organ specific inflammatory cells via different virulence factors such as its LPS and gingipains (How et al., 2016; Olsen et al., 2016). The bacteria, as well their molecules (capsular proteins, flagellin, fimbrillin, peptidoglycan, proteases), may be considered as pathogen-associated molecular patterns (PAMPs) and will interact with PRRs such as TLR-2 and TLR-4 resulting in pro-inflammatory cytokine secretion. This in turn could result in a neuroinflammatory state leading to neuronal destruction and the disruption of the blood–brain barrier (BBB) (Zlokovic, 2011; Keaney and Campbell, 2015). It is of note that P. gingivalis, as with all the above-mentioned microorganisms, is able to promote Aβ deposition, thus directly linking this infection to AD (Liu et al., 2017; Wu et al., 2017). These experimental data suggest that this is a physiological response of the organism to these microbiological challenges. Furthermore, P. gingivalis can disrupt the BBB (van de Haar et al., 2016), which may facilitate the entry of other cells and pathogens into the brain. Once more, the microorganisms of the mouth are also forming biofilms to assure their survival and their virulence (Liu et al., 2017) (immune evasion) and creating a quorum sensing like milieu which may affect the responses induced by each other (Srinivasan et al., 2017). This is of fundamental importance for their survival by protecting them from the host immune system. In summary, probably everybody would have P. gingivalis in their brains but depending on its virulence, on their genetic makeup and their susceptibility to develop inflammation, they may or may not be suffering from AD.

Another group of oral bacteria may also play a role in AD, namely Treponema, of which there are several species in the oral microbiome. Treponema pallidum, the infectious agent of syphilis, although not an oral treponeme, can invade the brain and provoke a chronic infection leading to neurosyphilis, which has common features with AD, due to its ability to evade the immune system. Oral treponemes have also been found in the brain and may also be able to efficiently evade the immune system and provoke chronic infections.

Recently, mutual influences in the gut–brain axis (bidirectional communication) have been suggested to contribute to the development of AD (Alkasir et al., 2017; Jiang et al., 2017). The microbiota are powerful modulators of whole-body metabolism. Dysbiosis, which can occur with aging, is associated with increased gut permeability that may influence several factors playing a role in the increased incidence of AD with age (Biagi et al., 2010). Most importantly, the dysregulated microbiota (gut, mouth, and nose) may lead to a systemic inflammatory state which impedes the functioning of brain cells including the activation of microglia at the origin of neuroinflammation.  Furthermore, this inflammation may also result in the invasion of microbes or microbial products such as LPS and amyloids into the brain (Bhattacharjee and Lukiw, 2013; Hufnagel et al., 2013), contributing to neuroinflammation and the resulting production of Aβ and pTau (Torrent et al., 2012; Schwartz and Boles, 2013; Hill et al., 2014; Bergman et al., 2016; Müller et al., 2017). Thus, it may be that changes occurring at different ages in the microbiome (dysbiosis) contribute to the development of AD.

Very recently, the involvement of fungi in AD has been demonstrated, following long-held suspicion that they may play a role (Pisa et al., 2016, 2017; Alonso et al., 2017). One of the most constantly mentioned mycetes is Candida albicans, which has been directly demonstrated in the brain of AD patients. Many other fungi have also been incriminated, the most frequent being the genus Malassezia. Malassezia are commensals of the oral cavity and also found on the skin. They may become pathogenic to humans in an opportunistic manner. So, most of the fungi originate from the nasal or the oral cavity. These disseminated mycoses may be implicated either as causative agents or as risk factors for AD. Interestingly, fungal products such as polyglucans, β-tubulin, enolase, and chitinase may also be found in the blood and the CSF from AD patients (Pisa et al., 2016). These are part of the body mycobiome (community of fungi inside an organism). However, it is important to mention that fungi are not only found in the brains of AD patients but also in normal and MCI brains. Thus, the question is once more how and why they may become pathogenic and lead to AD. Again, most probably genetic–environmental interactions will make them more or less virulent. Moreover, the number of fungi found, as well as their association with other microbes in senile plaques/biofilms may be important.

Even though there is very strong evidence that the brain of AD patients commonly contains several microbes this is clearly not enough to prove that AD is an infectious disease. It is now well-accepted that in the brain there is a large microbial biodiversity. Most of these organisms have been found in postmortem brains and documented in many countries throughout the world. So, this is a worldwide phenomenon. Also, this may emphasize the concomitant role of microbial products as triggers of the inflammatory status in the brain. One piece of the puzzle is still missing.

## NEW ROLE FOR Aβ

This missing link came from the seminal work of the group of Tanzi and Moir demonstrating that Aβ is an AMP (Soscia et al., 2010), as shown by comparing Aβ to LL37 (a powerful antimicrobial agent active against various bacteria and fungi). They tested Aβ as AMP against a large number of pathogens (e.g., Enterococcus faecalis, S. aureus, C. albicans, etc.). We found that Aβ also has antiviral activity, secreted upon viral infection with HSV-1 (Bourgade et al., 2015, 2016a,b). Indeed, Aβ creates pores in cellular membranes and thus kills bacteria, fungi, and enveloped viruses (Bode et al., 2017).

These observations were followed by the in vivo testing of the antimicrobial activity of Aβ. Mice infected with pathogens (e.g., Salmonella typhimurium) were intracerebrally treated with Aβ which almost totally prevented infection and plaque formation (Kumar et al., 2016). So, this confirmed the physiological antimicrobial activity of Aβ.

The demonstration that Aβ is an AMP was instrumental in making a link between the infection hypothesis of AD and the Aβ hypothesis of AD. This clearly indicates why trials that targeted Aβ failed and why Aβ, per se, could not be the cause of AD (Ferrer et al., 2004). This was followed by reassessment of the nature of senile plaques.

### NEW ASSESSMENT OF SENILE PLAQUES

In view of our infection hypothesis it can be said that the plaques in the brain of AD patients and even in the earlier stages may actually be biofilm.

A biofilm is a polysaccharide, protein, nucleic acid conglomerate secreted by one microorganism or by a synergistic microbial community (Costerton et al., 1995; Sapi et al., 2012; Serra et al., 2013; Kriebel et al., 2018). It surrounds the bacteria (or fungus) that secreted it and protects it from desiccation, toxic substances, antimicrobials, or immune attack. Within the biofilm, microorganisms can communicate via quorum sensing (Srinivasan et al., 2017). Many substances may be found inside the biofilm depending on the type of pathogen and the surrounding substances. Thus, microorganisms in biofilms show elevated tolerance to stress and antibiotics as well as to immune mediated attacks conferring to this whole structure an ideal niche to assure the persistence of the microorganism in the environment and mainly in association with the host.

When considering the infection hypothesis and the antimicrobial role of Aβ, it is suggested that senile plaque found in the brain is in fact a biofilm assuring the survival of various pathogens (polymicrobial). This was proposed by the group of Miklossy for treponema (Miklossy, 2016) and by the group of Balin for the C. pneumoniae (Balin et al., 2008). The group of Itzhaki also found evidence of HSV-1 DNA in plaques (Wozniak et al., 2009). Indeed, biofilms often contain microbial nucleic acids.

It is important to understand what the composition of senile plaque (biofilm) is. In biofilms, curli fibers, which are

microbial amyloids, aggregate and acquire Aβ-like conformations and act as cross-seeding molecules to propagate (Taylor and Matthews, 2015). Indeed, amyloid fibers are abundant in the bacterial world and are recognized as major structural and functional extracellular matrix components of environmental and pathogenic biofilms (Müller et al., 2017). Thus, it would be conceptualized that these microbial products which constitute the skeleton of the biofilm also incorporate Aβ in the brain which will result ultimately in senile plaques (Torrent et al., 2012; Bergman et al., 2016; Müller et al., 2017). As already mentioned, Aβ is an AMP so it is somehow counterintuitive to imagine that Aβ is used by the microorganism for its own destruction unless its incorporation into biofilm leads to its inactivation. In this case, it would not be surprising that microorganisms inactivate host antimicrobial agents. Alternatively, most research has mainly found residues of infectious agents but not live organisms in the AD brain. Thus, senile plaques may be a sort of cemetery for the various microorganisms, containing mainly very toxic and inflammatogenic microbial substances which may stimulate the surrounding microglia. However, it can be imagined that microorganisms may survive in some form that is not sensitive to Aβ attacks (perhaps within biofilms) allowing them to reactivate periodically.

Thus, presently all evidence converges to consider plaques as biofilms. If they may be found, as in some elderly, without dementia, this means that the biofilms may have efficiently contained the microorganisms and no clinical manifestations arise. When the microorganisms exit from the biofilms on periodic reactivation clinical symptoms may appear. These considerations make biofilms extremely important as targets or protectors against therapy. Nevertheless, despite the compelling evidence that senile plaques are biofilms this still does not definitively answer the fundamental question of whether microbes are the causative agents of AD.

### ROLE OF NEUROINFLAMMATION AND THE INNATE IMMUNE SYSTEM

Neuroinflammation is considered a hallmark of AD (McGeer and McGeer, 2013; Bolós et al., 2017; McManus and Heneka, 2017; Pritchard et al., 2017). The infection hypothesis provides the stimulus for this neuroinflammation. It also sheds light on two other fundamental issues: (1) Neuroinflammation may not be entirely the consequence of Aβ deposition (as stated by the amyloid hypothesis) but rather may itself cause Aβ deposition as a protective response toward microbial challenge; and (2) It is not the case that Aβ is exclusively a "harmful" molecule that aggregates to form plaque, but it is also a basic element of the innate immune defense system and thus a "beneficial" molecule as well, at least under certain conditions (Le Page et al., 2018). Ultimately, as reactivations of infections become more frequent and chronic production of Aβ increases, its antimicrobial effect may be blunted by loss of active Aβ through recruitment to plaque formation; inflammation becomes chronic and ultimately deposition proceeds and results in senile plaque formation (Bourgade et al., 2016a,b). The deposition of plaque may be the initiator of the inflammatory process, maintain it and finally destroy the neighboring neurons. This process becomes visible clinically when a threshold is crossed.

Thus, these considerations also suggest that the innate immune system plays an important role in the development and progression of AD not only in the brain but also outside the brain (Le Page et al., 2018). The infection will stimulate the innate immune system as participating cells join forces to eradicate the infections. There will be a successive activation of NK cells, neutrophils, and monocytes/macrophages all undergoing differential activation at different disease stages (from symptomfree to the MCI stage, then to fully developed AD) (Saresella et al., 2014; Le Page et al., 2015, 2017). Aβ is an actor but also a stimulus for the innate immune system, subsequently to other stimuli such as the microbial products found in the brain and in the periphery including LPS, curli products, glucans which act as PAMPs. They induce activation via PRR including TLRs and NODs resulting in a proinflammatory milieu with proinflammatory cytokines and chemokines, mainly TNFα, IL-1β, and IL-6 (McManus and Heneka, 2017). Indeed, an increased production of IL-1β and IL-18 by monocytes/macrophages was demonstrated after LPS stimulation reflecting the stimulation of the NLRP3 and caspase-1 pathways (Minogue, 2017; Wohleb and Delpech, 2017). Moreover, they can modify the phenotypes of the microglia as they transform them into M2 phenotypes. Thus, the innate immune system, by inducing neuroinflammation, plays a pivotal role in AD pathogenesis both in the periphery and after migrating to the brain through the increased BBB permeability.

### HOW MAY ALL THESE THEORIES BE RECONCILED?

The existing data suggest that AD results from a progressive accumulation of noxious inflammatory processes or events in the brain fuelled by multiple infectious agents that colonize/infect the body. Local neuroinflammation may continue at a low level throughout life with little negative effect. However, when exacerbated by reactivation of infections combined with other insults (e.g., oxidative stress), including age-related insults like increasing amounts of senescent cells, the acute inflammatory response results in unbalanced production of cytotoxic mediators, such as TNFα, which, accompanied by immunosenescence/inflamm-aging, becomes difficult to control or stop (Fulop et al., 2018). Microbial metabolites may not only fuel neuroinflammation, but also contribute to senile plaque formation when their biofilm components are integrated into plaque. The enhanced neuroinflammatory process damages neurons and alters the (BBB). These mediators also induce peripheral inflammation and then return to further stimulate local neuroinflammation (Blach-Olszewska et al., 2015; Festoff, 2016; Busse et al., 2017). This progressive proinflammatory situation is exacerbated with age, creating a vicious cycle of local and systemic inflammatory responses leading to activation of cytotoxic microglia, unbalanced cytokine production, Aβ accumulation and irreversible brain damage.

brain. This process leads to neuron death and eventually to AD.

### WHAT IS THE REQUIRED RESEARCH AGENDA TO TEST THE INFECTIOUS HYPOTHESIS?

We suggest that many microorganisms may together cause overproduction of Aβ by affected neurons while their biofilms enhance and reinforce senile plaque and thus they may be the actual cause of AD. By being able to detect them and their biofilms it should be possible to associate them with the development and progression of AD. An important question is how a chronic infection, potentially involving several microorganisms, may smolder for decades, its neuroinflammation remaining silent, and then manifesting itself clinically only decades later. Our preliminary results suggest that an infectious reservoir might exist inside the brain and/or in the periphery, which would be transmitted to the neurons. Reactivation of chronic infection may occur periodically under various stresses, but it is silenced by immunity until immune-surveillance is overcome or a threshold is reached. These chronic infections and reoccurrences would be favored by the presence of biofilms, which assure the survival of the infectious agent in the brain. Biofilm deposition in the brain may also contribute a framework for Aβ to create senile plaques. Indeed amyloid-like proteins made by bacteria form the basis of biofilms. Furthermore, polymicrobial biofilms are generally more robust than those made by individual bacteria and the addition of Aβ might make even stronger senile plaque/biofilm. Thus, detection of infection as early as possible, decades before the clinical manifestations may constitute a proof of infectious etiology.

### THERAPEUTIC IMPLICATIONS OF THE INFECTION HYPOTHESIS

It would be very optimistic to imagine that an antibiotic therapy may be administered and cure, or at least prevent progression of AD. It is clear, however that no antibiotic regimen has been developed to cure periodontitis which has a similar implication of chronic infectious microorganisms and biofilms. However, accepting that senile plaques have biofilm components opens avenues to different possibilities to attack the integrity of the biofilm. Small molecules, vaccines and other means could be developed to target the biofilms or the microorganisms that created them. It can be also imagined that by recognizing the infectious etiology of AD, we will be able to discover early markers of the disease and we could develop new prevention trials targeting new molecules. It is clear that Aβ should no longer be considered as the best or only promising target for the prevention and treatment of AD.

### CONCLUSION

fnagi-10-00224 July 20, 2018 Time: 14:59 # 8

Many experimental data support the involvement of a polymicrobial community in the pathogenesis of AD. This "neurobiome" would be the backbone to support the new infection hypothesis of AD (**Figure 1**). This new hypothesis naturally incorporates the old amyloid hypothesis as Aβ is again on the center stage but as a puppet to infectious masters, rather than as a principal actor. This also throws a new light on neuroinflammation which precedes Aβ production and is not a consequence of it. So, in our view the question is not whether the microorganisms are the causative agents of AD but how and when they are inducing it. How all these mostly commensal microorganisms became pathological and when in the evolution of the disease they will manifest themselves remains to be discovered. It can be assumed that advancing age may be a common driving factor to explain the how and the when by influencing either virulence factors or accumulation as well the progressively more senescent defense state against the infection which had installed itself perhaps decades earlier. This pathological situation is not without parallel to the natural history of cardiovascular diseases which also develop via atherosclerotic

### REFERENCES

lesions that appear decades before clinical manifestations. The age-associated loss of control of (neuro) inflammation will also play a role in AD. This new hypothesis generates hope to find new targets, even if biofilms are difficult to combat as reported in the case of periodontitis. Nevertheless, a better understanding of the role of the "neurobiome" will ultimately result in the prevention and treatment of this disastrous disease.

### AUTHOR CONTRIBUTIONS

TF, JW, KB, AK, EZ, AL, KH, GP, CB, GL, GD, and EF discussed and contributed to write the article. TF and EF conceptualized the article.

### FUNDING

This work was supported by grants from the Canadian Institutes of Health Research (CIHR) (Grant No. 106634), the Société des Médecins de l'Université de Sherbrooke, Program Audace of the Fond de Recherche Québec Société et Culture (Grant No. 2019-AUDC-263669) and the Research Center on Aging of the CIUSSS-CHUS, Sherbrooke, by the Polish Ministry of Science and Higher Education statutory (Grant No. 02-0058/07/262) to JW, and by the Agency for Science Technology and Research (A∗ STAR) to AL.

the fine regulation of T lymphocyte functions. Exp. Gerontol. 107, 161–168. doi: 10.1016/j.exger.2017.10.009

**Conflict of Interest Statement:** TF has received consultation fee from Eisai, Co.

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Fulop, Witkowski, Bourgade, Khalil, Zerif, Larbi, Hirokawa, Pawelec, Bocti, Lacombe, Dupuis and Frost. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# The Potential of LPS-Binding Protein to Reverse Amyloid Formation in Plasma Fibrin of Individuals With Alzheimer-Type Dementia

Etheresia Pretorius1 \*, Janette Bester2† , Martin J. Page1† and Douglas B. Kell1,3,4 \*

1 Department of Physiological Sciences, Faculty of Science, Stellenbosch University, Stellenbosch, South Africa, 2 Department of Physiology, Faculty of Health Sciences, University of Pretoria, Pretoria, South Africa, 3 School of Chemistry, The University of Manchester, Manchester, United Kingdom, 4 The Manchester Institute of Biotechnology, The University of Manchester, Manchester, United Kingdom

#### Edited by:

Judith Miklossy, Prevention Alzheimer International Foundation, Switzerland

#### Reviewed by:

Xinhua Zhan, University of California, Davis, United States Walter J. Lukiw, LSU Health Sciences Center New Orleans, United States

#### \*Correspondence:

Etheresia Pretorius<EMAIL_ADDRESS>Douglas B. Kell<EMAIL_ADDRESS>†Co-authors

Received: 15 January 2018 Accepted: 03 August 2018 Published: 22 August 2018

#### Citation:

Pretorius E, Bester J, Page MJ and Kell DB (2018) The Potential of LPS-Binding Protein to Reverse Amyloid Formation in Plasma Fibrin of Individuals With Alzheimer-Type Dementia. Front. Aging Neurosci. 10:257. doi: 10.3389/fnagi.2018.00257 Many studies indicate that there is a (mainly dormant) microbial component in the progressive development of Alzheimer-type dementias (ADs); and that in the case of Gram-negative organisms, a chief culprit might be the shedding of the highly inflammagenic lipopolysaccharide (LPS) from their cell walls. We have recently shown that a highly sensitive assay for the presence of free LPS [added to platelet poor plasma (PPP)] lies in its ability (in healthy individuals) to induce blood to clot into an amyloid form. This may be observed in a SEM or in a confocal microscope when suitable amyloid stains (such as thioflavin T) are added. This process could be inhibited by human lipopolysaccharide-binding protein (LBP). In the current paper, we show using scanning electron microscopy and confocal microscopy with amyloid markers, that PPP taken from individuals with AD exhibits considerable amyloid structure when clotting is initiated with thrombin but without added LPS. Furthermore, we could show that this amyloid structure may be reversed by the addition of very small amounts of LBP. This provides further evidence for a role of microbes and their inflammagenic cell wall products and that these products may be involved in pathological clotting in individuals with AD.

Keywords: Alzheimer-type dementia, amyloid, clotting, dormancy, infection, microbes

### INTRODUCTION

The progression of AD is accompanied by a great many observable changes, both molecular and physiological, and it is the commonest form of dementia (Takizawa et al., 2015). It is currently estimated that 5.4 million Americans have Alzheimer's Disease and that by mid-century the number of people living with Alzheimer's Disease in the United States alone is projected to grow to 13.8 million (Alzheimers Association, 2016).

We have previously shown that AD individuals present with various hematological abnormalities in terms of fibrin(ogen), platelet, and erythrocyte (RBC) structure, and this is summarized in **Figure 1**. In brief, AD individuals exhibit pathological levels of circulating cytokines, and "free" iron levels (albeit typically observed as serum ferritin) are also raised (Kell, 2009; Bester et al., 2013; Kell and Pretorius, 2014; Pretorius and Kell, 2014; Pretorius et al., 2016a). These circulating molecules are known to cause both hypercoagulation and hypofibrinolysis (Kell and Pretorius, 2015b). We have also suggested that, at least in part, the upregulation of cytokines and coagulation factors are due to the presence of potent circulating bacterial cell wall products, that include LPSs (Pretorius et al., 2016a). This purposely implies (as reviewed in Kell and Pretorius, 2018) that many of the pathologies seen in AD are due to the presence of the very potent circulating LPS inflammagen molecules (and other such molecules, e.g., lipoteichoic acid from Gram-positive bacteria). The presence of some sort of infection, with the infectious agents typically in a dormant state (Kell and Pretorius, 2015a; Kell et al., 2015; Potgieter et al., 2015), is central to this line of thought. It is supported by a great many papers that suggest that, although various risk factors have been identified and implicated in AD pathogenesis, including family history and genetics, central to the development of AD is in fact the presence of infections (e.g., Ripollés Piquer et al., 2004; Kamer et al., 2008a,b; Miklossy, 2008, 2011a,b; Honjo et al., 2009; Eriksson et al., 2011; Itzhaki and Wozniak, 2012; Amor et al., 2013; de Souza Rolim et al., 2014; Itzhaki, 2014; Karim et al., 2014; Shaik et al., 2014; Singhal et al., 2014; Singhrao et al., 2014; Gaur and Agnihotri, 2015; Itzhaki et al., 2016).

We recently reviewed the evidence that dormant, nongrowing bacteria are a crucial feature of AD, that their growth in vivo is normally limited by a lack of free iron, and that it is this iron dysregulation that is an important factor in their resuscitation (Potgieter et al., 2015; Pretorius et al., 2016a; Kell and Pretorius, 2018). We have also presented evidence that bacterial cells can be observed by ultrastructural microscopy in the blood of AD patients (Pretorius et al., 2016a). A consequence of this is that these bacterial cells might shed highly inflammatory components such as LPS. LPS is known to be able to induce (apoptotic, ferroptotic, and pyroptotic; Dong et al., 2015) neuronal cell death. LPS is also raised in AD, and it is found inside the brain and closely associated with the amyloid areas in the brains of these individuals (Lee et al., 2008; Deng et al., 2014; Zhao and Lukiw, 2015; Zhao et al., 2015). Recently, Zhan and co-workers also reviewed literature showing that Gram-negative bacteria (E. coli) can induce the formation of extracellular amyloid, and that the degraded myelin basic protein (dMBP) colocalizes with β amyloid (Aβ) and LPS in amyloid plaques in AD brains (Zhan et al., 2018).

We recently also provided evidence that LPS (and LTA from Gram-positive bacteria) could induce amyloid formation in healthy fibrin(ogen), the most abundant plasma protein in blood, after it is added at tiny concentrations to blood from healthy individuals (followed by the clotting agent thrombin) (Pretorius et al., 2016b, 2018a). We then studied the presence of amyloid in these clots (before and after addition of LPS), using confocal microscopy and fluorescent markers for amyloid. In those experiments, we saw that addition of LPS to healthy PPP caused a significant increase of amyloid fluorescent signal, compared to the naïve sample (i.e., samples without added LPS). In these papers, we also showed that LBP can inhibit the formation of such amyloid structures (Pretorius et al., 2016b, 2018a). Furthermore, we showed that (some) of the (naïve) fibrin(ogen) molecules are amyloid in conditions such as type 2 diabetes and Parkinson's Disease, and that in these conditions, LBP added to PPP of such individuals, could also reduce the extent of amyloid fibrin(ogen) structure (Pretorius et al., 2017a,b, 2018a,b).

Thus, the question now arose as to whether the extent of fibrin-type amyloid in PPP varies between AD individuals and suitably matched controls, and whether the removal of any LPS using the mopping agent, LBP, could remove the amyloid signal present in the (naïve) plasma of AD individuals.

Indeed, Zhang et al. (2009) reported elevated levels of LPS concentrations in plasma from patients with sporadic amyotrophic lateral sclerosis and AD, as compared to healthy controls. The present paper provides further evidence of the presence of LPS in PPP of AD individuals, as we showed that LBP could remove amyloid (fluorescent) signal from AD plasma. Our observation is therefore consistent with the general view set out above that there is a major dormant microbial component to AD.

### MATERIALS AND METHODS

### Ethical Statement, Volunteer Details, and Blood Collection

Blood samples were obtained from non-smoking, Alzheimertype dementia (AD) patients, identified by a Neurologist and under the care of a medical practitioner. Specifically, care was taken to exclude vascular dementia. We also recruited "healthy" age-matched individuals that did not smoke. It should be noted that the term "healthy" is used in this paper to describe an individual who does not have dementia. Ethical clearance was obtained from the Health Sciences Ethical committee from the University of Pretoria, and informed consent was obtained from family members who act as carers of the patients (81/2013, amended 2015). Healthy individuals also filled in consent forms. Blood was collected in two 4 mL citrate tubes and one 4 mL clotting tube for iron level determination. This collection and all handling of samples were performed under very strictly aseptic conditions, to prevent any microbial contamination of samples.

### Iron Tests

Serum ferritin, transferrin, and serum iron was tested at a pathology laboratory in South Africa.

**Abbreviations:** AD, Alzheimer-type dementia; LBP, lipopolysaccharide-binding protein; LPS, lipopolysaccharide; PPP, platelet poor plasma; SEM, scanning electron microscope.

### LPS-Binding Protein

fnagi-10-00257 August 22, 2018 Time: 16:22 # 4

A final added LBP exposure concentration of 4 ng L−1 LBP was used and LBP was purchased from Sigma (recombinant product SRP6033; >95% pure).

### Scanning Electron Microscopy (SEM) of Platelet Poor Plasma (PPP)

At least 30 min after the blood was collected in citrate tubes by venepuncture, PPP were obtained and frozen at −80◦C. PPP was prepared by centrifuging citrated whole blood for 15 min at 3,000 g at room temperature. After all samples were collected, PPP were thawed and 10 µL mixed with 5 µL thrombin to create an extensive fibrin network. Thrombin was provided by the South African National Blood Service, and the thrombin solution was at a final exposure concentration of 10 U mL−1 (initial product concentration is 20 U mL−1 made up in PBS containing 0.2% human serum albumin, see footnote 1 for a description of how thrombin units are calculated). A Zeiss ULTRA Plus FEG-SEM with InLens capabilities was used to study the surface morphology of erythrocytes, and micrographs were taken at 1 kV. SEM preparation was done as previously reported (Pretorius et al., 2017c).

### Airyscan Confocal Microscopy

PPP was thawed, followed by preparation of clots for analysis using confocal Airyscan methods. We added Thioflavin T (ThT) (a well-established amyloid stain; LeVine, 1999; Biancalana et al., 2009; Biancalana and Koide, 2010; Groenning, 2010; Sulatskaya et al., 2011, 2012; Kuznetsova et al., 2012; Picken and Herrera, 2012; Younan and Viles, 2015; Kuznetsova et al., 2016; Rybicka et al., 2016) at a final concentration of 5 M to 200 µL to either healthy PPP, naïve AD PPP, or after a 10 min exposure of AD PPP to 4 ng L−1 (final concentration) LBP. These PPP samples were incubated (protected from light) for 1 min. This step was followed with the addition of thrombin, added in the ratio 1:2 to create extensive fibrin networks. A coverslip was placed over the prepared clot, and viewed immediately with a Zeiss LSM 510 META confocal microscope with super-resolution (Airyscan) capabilities. The Airyscan detector increases the resolution by a factor of 1.7, achieving super-resolution of 140 nm, and with a Plan-Apochromat 63×/1.4 Oil DIC objective. Excitation was at 488 nm and emitted light was measured at 505–550 nm.

## Statistical Analysis and Data-Sharing

Histogram-Based Analysis of SEM and ThT Staining

For each picture, we obtained the histogram of intensities (8 bit scale) using the histogram function of ImageJ. From this we calculated the coefficient of variation (CV; as standard deviation/mean). For details of this analysis method, see (Pretorius et al., 2017b, 2018a). Quantification of fluorescent marker binding (ThT) was done by assessing the variance between (black) background and the presence of fluorescent pixels where ThT fluorescent binding was present in the clots. Increased ThT binding is here reflected as increased fluorescence which shows increased amyloid protein structure in fibrin(ogen) (see Pretorius et al., 2017a, 2018a) for a detailed explanation of the methods. We used the histogram function in ImageJ (FIJI) and calculated the coefficient of variation (CV) (as SD/mean) of the histogram of different pixel intensities as our metric to quantify and discriminate between clots of healthy (age-controlled) naïve PPP and clots from AD with and without LBP.

A healthy clot (i.e., a clot taken from a healthy individual), viewed with SEM looks somewhat like a bowl of spaghetti with elongated fibrin fibers. In AD individuals, this clot structure changes to a dense and matted hypercoagulated clot (Bester et al., 2015). We also used the CV calculation described above to analyze SEM clots. The fibrin fibers of healthy individuals have a greater variation of dark and light areas, due to the elongated fibers, with open areas between the individual fibers. With an increased hypercoagulability and amyloid formation, the clots become matted and dense, resulting in a more uniform grayness. We used this difference in structure as our metric, where increased hypercoagulability is related to an increase in amyloid formation and this is visible as a more uniformly dense morphology with less color gradient.

The statistical analysis of CV data was performed with GraphPad 7, using the one-way ANOVA analysis with Tukey's multiple comparison's test comparing the mean of each column with the mean of every other column.

### Availability of Data and Material

Raw data, including original micrographs can be accessed at: https://1drv.ms/f/s!AgoCOmY3bkKHiJRrop6cF6uhTnQA1A or https://www.researchgate.net/profile/Etheresia\_Pretorius.

### RESULTS

As discussed in the introduction, AD is not only known for the presence of neuroinflammation, but also for the presence of hematological abnormalities, including an increased presence of LPS and also (dysregulated) cytokines, iron and clotting factors, which result in oxidative stress and abnormal clotting. Previously we showed that abnormal clotting and the presence of bacterial inflammagens like LPS, result in fibrin(ogen) becoming amyloid in nature, and that we can remove the signal by addition of LBP (Pretorius et al., 2017a,b, 2018b). In **Figure 1**, we set out our hypothesis: that also in AD, the presence of LPS, together with dysregulated iron levels and oxidative stress, causes fibrin(ogen) to become amyloid and that we can reverse this with LBP. Furthermore, we show this reversal by using both ultrastructure (SEM) and the fluorescent marker ThT using Airyscan (confocal) microscopy. The rationale behind using LBP is that, if the amyloid structure is indeed due to the presence of bacterial inflammagens, LBP would remove it by binding to these inflammagnes, thus preventing it from causing amyloid fibrin(ogen) deposits.

**Table 1** shows the demographics of individuals with AD, as well as healthy, age-controlled individuals. Transferrin, iron, % saturation of iron and serum ferritin were measured in these

1https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/ Product\_Information\_Sheet/1/t6884pis.pdf

Gender was compared using Fisher's exact test. Age and iron measurements were compared using the unpaired t-test. Serum ferritin was compared using the Mann– Whitney test following the non-normal distribution of this measurement. All analyses were done using GraphPad 7. Data are presented as either mean ± STD or median (lower quartile–upper quartile; interquartile range). Bold numbers show significant p-values.

individuals, and these values, particularly serum ferritin, is used as an indication of the level of systemic inflammation (Kell, 2009; Kell and Pretorius, 2014).

In our hypothesis and **Figure 1** we argue that there is a link between oxidative stress, increased iron levels and inflammation, and this is directly linked to the presence of bacterial inflammagens like LPS. In our sample, healthy individuals had low mean serum ferritin, where in the AD population it was approximately three times higher. However, despite the large difference in mean serum ferritin values between the two groups, the difference was not statistically significant owing to large variation within the samples.

**Table 2** shows results for the analysis of the clots using both SEM and confocal microscopy. Micrographs were analyzed as discussed in Section "Materials and Methods". **Table 2** shows p-values and statistics of CVs calculated from SEM (micrographs showing ultrastructure) and Airyscan (micrographs showing fluorescence). We compared CVs from

TABLE 2 | Data for Alzheimer-type dementia and healthy individuals showing the coefficients of variation (CV) of the intensity of the pixels in the clot images (Tukey's analysis).

Airyscan and SEM images were used and statistical analysis was done to compare CVs from controls vs. AD individuals. Bold numbers show significant p-values.

controls and AD individuals, and that produced the p-values (**Table 2**).

**Figure 2A** gives an example of the clot structure, as viewed with SEM, from a representative healthy individual. We analyzed each SEM micrograph with ImageJ and produced a histogram that gave us the mean and the standard deviation for each micrograph (see section "Materials and Methods"). **Figure 2B** shows such a representative histogram of the 8-bit intensity for the SEM micrograph shown in **Figure 2A**. All micrograph histograms were used to calculate the CVs for each participant (both controls and AD individuals) (statistical analysis shown in **Table 2**). **Figure 3** shows SEM images before and after treatment of a representative examples of three AD PPP clots, with and without LBP.

**Figure 4** show a representative micrograph and its histogram from a healthy individual, using Airyscan confocal microscopy. **Figure 5** shows clots from AD individuals before and after LBP treatment. In healthy clots, there is little to no binding of ThT to amyloid fibrin(ogen) proteins. In AD clots, significant

with SEM. All clots were created by adding thrombin to PPP (prepared after whole blood is centrifuged for 15 min at 3,000 g). (B) Representative histogram of the 8-bit intensity for the SEM clot shown in (A).

ThT binding fluorescence is noted, suggesting increased amyloid formation in fibrin(ogen). When LBP is added to AD PPP, ThT show significantly decreased binding. **Figures 6A,B** show graphs and boxplots from the CV analysis. LBP added to PPP from AD individuals (with added thrombin to initiate clotting), seems to aid in the removal of amyloid signal so that the fibrin(ogen) structure now looks more like that of the controls (noted by using two techniques: Airyscan and SEM). Furthermore, the p-values between controls vs. AD with added LBP in both the

Airyscan and SEM analysis, showed that added LBP makes AD clots not significantly different to the controls (p = 0.8 and 0.06).

opposite column (A), after treatment with LBP, followed by addition of ThT

## DISCUSSION

and clot preparation.

We have previously determined that in many inflammatory conditions, the "normal" clotting of blood, involving the polymerisation of fibrinogen to fibrin, produces a fibrin fiber structure that becomes amyloid in nature, and that this might be due to the presence (in part) of the potent inflammagen LPS, which comes from the membranes of Gram-negative bacteria (Potgieter et al., 2015, 2016b, 2017b, 2018a; Kell and Pretorius, 2017a,b) and is a potent inflammagen (Walter et al., 2007; Kell and Pretorius, 2015a). This would be consistent with the many studies (reviewed in Miklossy, 2015; Itzhaki et al., 2016; Kell and

Pretorius, 2018) that imply that there is a (dormant) microbial component in AD. Previous research (see Poole et al., 2013; Bester et al., 2015; Zhan et al., 2016a,b; Zhao et al., 2017a,b) found LPS inside the brains of Alzheimer's disease patients, as well as an increase in circulating LPS. LPS is known to cross (and possibly to damage Liu et al., 2001; Xaio et al., 2001; Jaeger et al., 2009; Jangula and Murphy, 2013; Banks et al., 2015) the BBB and lead to β-amyloid depositions (Lee et al., 2008). Furthermore, neurotoxic microbial-derived components from the GI tract microbiome can cross aging GI tracts and BBBs and contribute to progressive proinflammatory neurodegeneration (Zhao and Lukiw, 2018). In a recent review, Zhan and coworkers describe that LPS indeed associates with amyloid plaques, neurons and oligodendrocytes in AD brains (Zhan et al., 2018). These authors also showed that LPS infiltrates the AD nucleus and can induce an inflammatory signaling program in brain cells, including up-regulation of the pro-inflammatory microRNA miRNA-146a via a NF-kB signaling circuit (Zhan et al., 2018).

Here we also show that the hypercoagulable structure of fibrin(ogen) in AD patients is different from healthy individuals, in that they appear to be amyloid (as shown with the fluorescent marker ThT) and that their structure, viewed with SEM, is matted and dense. In healthy clots, fibrin has a typical "spaghetti-like" structure (Kell and Pretorius, 2015b). We could reverse aberrant clotting in AD PPP by the addition of LBP. LBP binds bacterial inflammagens and our results would therefore point to the presence of bacterial inflammagens in AD PPP – that is, LBP could bind to and thus prevent these inflammagens from causing amyloid formation in the AD PPP when clots are formed after addition of thrombin.

When we added LBP to PPP from AD individuals (by incubating their PPP with LBP), we showed that the p-values were not significantly different (p = 0.8 and 0.06) between AD and control donor blood. Therefore, LBP, incorporated in a therapy, might not only prevent aberrant clotting in these individuals, but might also reduce the circulating LPS pool that could eventually cross into their brains via the BBB. Of course, a damaged BBB can admit the transfer (atopobiosis; Potgieter et al., 2015) of the organisms themselves (Miklossy, 2011a, 2015; Bajpai et al., 2014; Tang et al., 2017), where they may be detected ultrastructurally (Mattman, 2001), and that may continue to shed inflammagens. We therefore suggest that LBP might eventually be used as treatment to prevent the damaging effect of LPS on fibrin(ogen) and hypercoagulation, and even to prevent (at least in part) the deposition of amyloid-β (Aβ) plaques in the brain and the loss of cognitive function that accompanies this neurodegenerative disease. However, we note that a control protein, such as human IgG should, in future, be used to present the specific effect of LBP on amyloid formation, to further elucidate the physiological processes discussed in this paper. In future, our hypothesis could also be tested in a transgenic murine model of AD (TgAD) or the 5xFAD (amyloid over-producing) model or equivalent (Vale et al., 2010; Jeong et al., 2018).

### REFERENCES

### AUTHOR CONTRIBUTIONS

EP study leader, prepared all the figures, and co-wrote the paper. JB prepared and analyzed all the samples. MP statistical analysis and the paper editing. DK study co-leader, and co-wrote and edited the paper. All authors reviewed the manuscript.

### FUNDING

We thank the Biotechnology and Biological Sciences Research Council (Grant No. BB/L025752/1) as well as the National Research Foundation (NRF) of South Africa (91548: Competitive Program) and the Medical Research Council of South Africa (MRC) (Self-Initiated Research Program) for supporting this collaboration.

### ACKNOWLEDGMENTS

This is paper 16 in the series "a dormant blood microbiome in chronic, inflammatory diseases." The authors thank Dr. Prashilla Soma: Clinician.

**Conflict of Interest Statement:** The authors (DK and EP) declare the following patent application: method for treating Alzheimer's Disease (P3448ZA00-AS2CA).

The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2018 Pretorius, Bester, Page and Kell. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

# Chlamydia pneumoniae: An Etiologic Agent for Late-Onset Dementia

Brian J. Balin1 , Christine J. Hammond1 , Christopher Scott Little1 , Susan T. Hingley 1 , Zein Al-Atrache1 , Denah M. Appelt 1 , Judith A. Whittum-Hudson2 and Alan P. Hudson2 \*

1Department of Bio-Medical Sciences, Center for Chronic Disorders of Aging, Philadelphia College of Osteopathic Medicine, Philadelphia, PA, United States, 2Department of Biochemistry, Immunology and Microbiology, Wayne State University School of Medicine, Detroit, MI, United States

The disease known as late-onset Alzheimer's disease is a neurodegenerative condition recognized as the single most commonform of senile dementia. The condition is sporadic and has been attributed to neuronal damage and loss, both of which have been linked to the accumulation of protein deposits in the brain. Significant progress has been made over the past two decades regarding our overall understanding of the apparently pathogenic entities that arise in the affected brain, both for earlyonset disease, which constitutes approximately 5% of all cases, as well as late-onset disease, which constitutes the remainder of cases. Observable neuropathology includes: neurofibrillary tangles, neuropil threads, neuritic senile plaques and often deposits of amyloid around the cerebrovasculature. Although many studies have provided a relatively detailed knowledge of these putatively pathogenic entities, understanding of the events that initiate and support the biological processes generating them and the subsequent observable neuropathology and neurodegeneration remain limited. This is especially true in the case of late-onset disease. Although early-onset Alzheimer's disease has been shown conclusively to have genetic roots, the detailed etiologic initiation of late-onset disease without such genetic origins has remained elusive. Over the last 15 years, current and ongoing work has implicated infection in the etiology and pathogenesis of late-onset dementia. Infectious agents reported to be associated with disease initiation are various, including several viruses and pathogenic bacterial species. We have reported extensively regarding an association between late-onset disease and infection with the intracellular bacterial pathogen Chlamydia pneumoniae. In this article, we review previously published data and recent results that support involvement of this unusual respiratory pathogen in disease induction and development. We further suggest several areas for future research that should elucidate details relating to those processes, and we argue for a change in the designation of the disease based on increased understanding of its clinical attributes.

Keywords: late-onset dementia, Alzheimer's disease, amyloid, APOE, Chlamydia pneumoniae, etiology, infection, neuroinflammation

### INTRODUCTION

A longstanding idea in the medical literature is that a wide variety of chronic diseases could be caused or exacerbated by a microbial infection. For example, in the early 20th century rheumatoid arthritis was considered to be an infectious disease, an explanation that was more or less abandoned by mid-century but which has re-emerged (Lansbury, 1950; Ford, 1963;

#### Edited by:

Judith Miklossy, Prevention Alzheimer International Foundation, Switzerland

#### Reviewed by:

Michael Lardelli, University of Adelaide, Australia Robert V. Schoborg, East Tennessee State University, United States

> \*Correspondence: Alan P. Hudson<EMAIL_ADDRESS>
Received: 19 March 2018 Accepted: 13 September 2018 Published: 09 October 2018

#### Citation:

Balin BJ, Hammond CJ, Little CS, Hingley ST, Al-Atrache Z, Appelt DM, Whittum-Hudson JA and Hudson AP (2018) Chlamydia pneumoniae: An Etiologic Agent for Late-Onset Dementia. Front. Aging Neurosci. 10:302. doi: 10.3389/fnagi.2018.00302 Albert, 2000; Carty et al., 2004). Multiple sclerosis long has been attributed at least in part to involvement of an infectious component, although the identity of the specific agent(s) remain(s) to be firmly established (Swanborg et al., 2003). Similar arguments have been made in regard to other chronic diseases of an idiopathic nature, but in most cases, an infectious etiology and/or involvement of microbes in disease exacerbation have been difficult to establish. The pattern is paralleled in cancer etiology, in which infectious causation of some cancers has been demonstrated (for review see de Martel et al., 2012); however, most cancers are not thought to have an infectious etiology.

Instead of viral, bacterial, or mycological involvement in disease causation, alternative mechanisms that might explain chronic disease genesis have been pursued. Among the most prominent explanations has been the potential genetic basis of chronic clinical entities, including what is termed late-onset Alzheimer's disease. Most of these studies have indicated that development of disease is not easily attributable to one or even a few mutations or gene polymorphisms. Rather, such studies have indicated that disease genesis is multifactorial, resulting from as yet unknown environmental and host genetic factors (Harney and Wordsworth, 2002; O'Connor et al., 2006).

With regard to Alzheimer's disease, the single idea that has predominated for almost three decades in studies of the neuropathology underlying this clinical entity is the ''amyloid cascade'' hypothesis. The hypothesis proposes that the deposition of the amyloid-β (Aβ) peptide is the critical event underlying neuronal degeneration, and thus cognitive dysfunction (Schellenberg, 1995). This idea is appropriately applicable to the early form of dementia, familial Alzheimer's disease, since it is well established that this type of dementia is caused by genetic mutations resulting in an increase in amyloid formation and deposition (Tanzi, 2012). However, many studies have determined that causation of late-onset disease is not attributable to a small number of identical, similar, or other genetic defects leading to Aβ deposition, thereby undermining the contention that Aβ is the universal etiologic factor eliciting the dementia. Importantly, in both familial Alzheimer's disease and late-onset disease, the neuropathology is essentially identical; this clearly indicates that factors other than the genetic lesions which underlie familial disease must exist to explain the neuropathology of late-onset disease.

Late-onset dementia typically is observed in older age; indeed, age is the primary risk factor for its development. Other factors also may increase risk of late-onset disease.  Intriguingly, many of the same risk factors could promote systemic inflammation that may also influence disease pathogenesis. Furthermore, chronic, persistent, or latent infections in the brain, all of which may be an outcome following infection with a variety of organisms (see Itzhaki et al., 2004), could reactivate to initiate and/or exacerbate late-onset disease. These possibilities implicate both systemic and neuroinflammation in late-onset dementia. Thus, a likely scenario for development of late-onset dementia centers on poorly understood interactions between genetic risk, as exemplified in part by possession of the Apolipoprotein E (APOEε)4 allele, and environmental factor(s), including infection. For these and other reasons, we have argued that late-onset disease is distinguishable from the genetically-based early-onset disease. On that basis we suggested the designation ''late-onset dementia of the Alzheimer's type'' for the late-onset clinical entity, since it is more consistent with our current knowledge of the clinical and neuropathological underpinnings of the disease (Balin et al., 2017); in this article, we employ that designation, or simply late-onset dementia, throughout.

Many attempts have been made to identify infectious agents responsible for late-onset dementia of the Alzheimer's type (see e.g., Emery et al., 2017). However, none of the agents studied has been unequivocally accepted to be either etiologic, or indeed exacerbating, for dementia-related neuropathology. These include many viral pathogens such as measles virus, lentiviruses, adenovirus and others (Pogo et al., 1987; Friedland et al., 1990). Importantly, studies of herpes simplex virus type 1 (HSV-1) infection in late-onset disease identified this virus as a risk factor for people expressing APOEε4 (Itzhaki et al., 1997, 2001). Further, bacterial pathogens, including Chlamydia trachomatis, Coxiella burnettii and others have been investigated, but no clear relationship with Alzheimer's pathogenesis has been demonstrated to date (Renvoize et al., 1987). In contrast, we discovered an association between the genesis of late-onset disease and infection with the intracellular respiratory bacterial pathogen Chlamydia pneumoniae. (Balin et al., 1998; Gérard et al., 2006). We summarize that work in this review, along with recently published studies from our group and those of others' that implicate involvement of this unusual and ubiquitous pathogen in the genesis of senile cognitive dysfunction reflective of late-onset disease.

### CHLAMYDIA PNEUMONIAE

C. pneumoniae, an obligate intracellular bacterium, is a pathogen of the respiratory tract, infecting mucosal surfaces, specifically the lung/pulmonary and nasal mucosa (Grayston et al., 1990; Campbell and Kuo, 2002; Hahn et al., 2002). It is ubiquitous in all societal cultures and geographic regions studied to date (Leinonen, 1993). Many reports have demonstrated that the organism is responsible for a significant proportion of community-acquired pneumonia, and it has been associated with numerous other pulmonary diseases (Grayston et al., 1990; Clementsen et al., 2002). Interestingly, C. pneumoniae infections also have been linked with an array of non-respiratory diseases, including atherosclerosis, inflammatory arthritis, multiple sclerosis and others (Sriram et al., 1998; Schumacher et al., 1999; Wagner et al., 2000; Belland et al., 2004). While some such associations are certainly controversial, credence has been gained for the role of this organism in atherogenesis during the last 20 years (Grayston, 2000; Rosenfeld et al., 2000; Belland et al., 2004).

Similar to other chlamydial species, C. pneumoniae displays a biphasic developmental cycle. The first phase involves the infectious extracellular form of the organism, the elementary body (EB). EB typically infect epithelial cells, but many cell types can be infected, including cells in the nervous system such as astroglia, microglia and neurons (Hatch, 1999; Dreses-Werringloer et al., 2006; Gérard et al., 2006). Following endocytosis into a cytoplasmic inclusion, EB reorganize into a reticulate body (RB), which is the metabolically active form of the organism. RB undergo multiple cycles of cell division followed by a ''dedevelopmental'' process yielding the infectious EB form again. These are released via eukaryotic cell lysis or exocytosis, which spreads the infection (Hatch, 1999).

Systemic dissemination of the organism from the respiratory tract has been well-documented (Gieffers et al., 2004), and several studies have indicated that this occurs via monocytes (Moazed et al., 1998). Importantly, C. pneumoniae, like other chlamydial species, undergoes long-term infection in a biological state termed ''persistence'' (Hogan et al., 2004). In persistence, the organism, as with other chlamydial species, is viable and metabolically active but does not complete its normal developmental cycle. Further, persistent chlamydial infections have been shown to be refractory to antibiotic treatment (e.g., Deniset and Pierce, 2010; Phillips-Campbell et al., 2014).

### INITIAL STUDIES OF C. PNEUMONIAE AND LATE-ONSET DISEASE

Our initial studies of possible involvement of C. pneumoniae in late-onset disease identified DNA of the organism in 90% of postmortem brain samples from patients diagnosed with late-onset dementia of the Alzheimer's type; this study employed highly specific polymerase chain reaction (PCR) assays (Balin et al., 1998; Schumacher et al., 1999). Only 5% of control non-demented brain samples contained C. pneumoniae DNA. Positive DNA results were obtained from brain tissues from areas that normally display characteristic neuropathology (e.g., hippocampus) and from those less often demonstrating characteristic pathology (e.g., cerebellum). In nearly 90% of affected brain samples, positive PCR signals were obtained from at least one area showing neuropathology, and from the cerebellum in four cases. In these four cases, neuropathology existed within the cerebella as well as other areas.  We further analyzed frozen brain samples for intact bacterial RNA using reverse transcriptase-PCR (RT-PCR). mRNA species encoding the KDO transferase and a ∼376 kDa protein specific to C. pneumoniae were successfully targeted.

In these initial studies, immunohistochemistry and electron microscopy also were used to analyze samples from PCR-positive brains. Samples from individuals with clear late-onset disease contained C. pneumoniae antigens, specifically in cortical regions such as the temporal, parietal and pre-frontal cortices as well as the hippocampus. In these regions, organism immuno-positivity was observed in various cell types including perivascular macrophages, microglia and astroglia. Ultrastructural analysis of the positive brain samples revealed chlamydial inclusions containing both EB and RB. Immuno-electron microscopy using a gold-conjugated monoclonal antibody specific to an outer membrane protein demonstrated gold particles labeling the organism (Balin et al., 1998; Arking et al., 1999). Immunoelectron microscopy was negative in comparable PCR-negative control brain sections.

PCR and RT-PCR positive sample homogenates were prepared and incubated with human THP-1 monocytes in culture. Viable bacteria were successfully recovered from two brains positive for the organism. Culture of homogenates from two control brains prepared in a similar fashion generated negative results (Balin et al., 1998). Thus, DNA and antigens of C. pneumoniae were found in areas of neuropathology in brain samples from late-onset disease individuals, and samples from frozen brain tissues from these subjects yielded viable organisms. Genetic analyses revealed that 11 of the 17 PCR-positive samples had at least one allele for the APOEε4 isoform (64%), consistent with that allele type being a risk factor for development of late-onset dementia (Roses, 1996). Importantly, in a separate study, it was shown that in patients with reactive arthritis who had DNA from C. pneumoniae in synovial tissues, 2/3 had one or more copies of the APOEε4 allele (Gérard et al., 1999). Together, these and other observations discussed below clearly implicate a relationship between the APOEε4 allelic genotype and infection by C. pneumoniae; they suggest that both factors confer an increased risk for chronic disease genesis (Balin et al., 1998; Gérard et al., 1999).

Not surprisingly, these initial studies and their implications suggesting that an infection with a common bacterium is involved in the origin of late-onset dementia instigated several other groups to attempt confirmation of the organism in brain tissues from other affected and control patient cases. Those studies yielded mixed results, with some reports providing positive identification (e.g., Mahony et al., 2000; Ossewaarde et al., 2000; Di Pietro et al., 2013a), and others failing to find DNA or antigens from the organism in relevant samples (e.g., Nochlin et al., 1999; Gieffers et al., 2000; Ring and Lyons, 2000; Taylor et al., 2002). Importantly and as reviewed by us previously in detail, numerous and various different techniques were used in the confirmation studies, with none utilizing methodology identical to our own. For example, one study failed to find C. pneumoniae in paraffin-embedded brain tissues from confirmed late-onset dementia patients via PCR or immunohistochemical (IHC) analyses (Nochlin et al., 1999); similarly, the organism was not found in several relevant paraffin-embedded brain samples using either PCR or IHC (Gieffers et al., 2000). The recovery of reasonable quality template for PCR from paraffin-embedded tissue can be unreliable, which might explain some negative results. We used exclusively frozen brain-tissue samples were in our studies. One report indicated success in identifying C. pneumoniae by in situ hybridization in many relevant brain samples (Ossewaarde et al., 2000); controls were negative, including those from individuals with other neurological diseases. In our studies, in negative IHC studies, and in the positive study, the same mAb targeting the C. pneumoniae MOMP or lipopolysaccharide (LPS) were employed, suggesting differences in technique in obtaining positive results. Another report explains the positive and negative PCR data from some studies (Mahony et al., 2000). This study employed replicate PCR assays and probit regression analyses to show that DNA from most frozen brain samples analyzed from relevant patients was PCR-positive for C. pneumoniae if enough replicates were performed; multiple assayed controls were always PCR-negative. Clearly, discrepancies in analytical methods used among the different laboratories severely constrain the results obtained (Campbell and Kuo, 2004).

### Confirmatory Studies

In subsequent research to confirm and extend our initial studies, we analyzed new tissue samples from late-onset dementia and control brains (Gérard et al., 2006). PCR analyses targeting two C. pneumoniae genes showed that samples from 20/25 late-onset brains, but only 3/27 control brains, were PCR-positive (Gérard et al., 2006). Viable organisms were cultured from the former brain samples, and metabolic activity of the cultured bacteria was demonstrated via identification of several intact chlamydial transcripts. Immunohistochemistry of relevant brain samples identified that astroglia, microglia and ∼1/5 of neurons were infected with C. pneumoniae. As in our initial study, infected cells were located in the brain in proximity to both senile plaques and nerve cells containing neurofibrillary tangles (Balin et al., 1998). Together, these observations suggested that C. pneumoniae infection had a direct effect on neuronal cell injury/death. Further, the potential for the organism to act as initiator of granulovacuolar degeneration, another previously acknowledged pathology in late-onset disease, was suggested (Funk et al., 2011).

Continued analyses demonstrated that immunolabeling was positive for C. pneumoniae in the entorhinal and frontal cortices, and in the hippocampal formation of all relevant affected brains (Hammond et al., 2010). These areas exhibited both amyloid deposition and Chlamydia immunoreactivity in apposition to one another when stained with Thioflavin S and labeled with anti-C. pneumoniae antibodies on the same sections, thus revealing fibrillary amyloid and chlamydial immunoreactivity, respectively. Two extracellular patterns of chlamydial immunoreactivity were observed: a punctate pattern and a pattern with amorphous foci. These may represent extrusion of whole organism (punctate) or secreted chlamydial products, e.g., LPS (amorphous foci), into the tissues (Stuart et al., 1994; Hybiske and Stephens, 2007). Amyloid is known to have anti-microbial properties, perhaps allowing it to act as an anionic defensin (Bishop and Robinson, 2004; Kammerman et al., 2006; Soscia et al., 2010; Kumar et al., 2016). As reflected by the deposition of amyloid in the same region as C. pneumoniae, tropism of this organism to the central nervous system (CNS) may be a precursor or trigger for development of damage (Hammond et al., 2010).