Disease Burden

Alzheimer’s Disease is the 6th leading cause of death in the U.S. with approximately 5.7 million Americans currently living with this devastating disease.  By 2050 the number is projected to rise to 14 million people.  The financial burden of Alzheimer’s and Dementia is astounding.  It will cost our nation roughly $277 billion in 2018.  To date, ineffective therapies targeting Amyloid Beta highlight the need for innovation.  At Intracell Research Group we believe that a significant subset of dementia and Alzheimer’s patients are experiencing a “syndrome” being driven by one or more infectious pathogens.   Chlamydia pneumonaie, Herpes Viruses, and Spirochetes have all been published in correlation to the onset and progression of Dementia/Alzheimer’s.  Amyloid Beta has been identified as the bodies response to infection.  It exhibits antibacterial and antiviral activity.  This and several other clues substantiated by the research below are in desperate need of attention.  We advocate a randomized, controlled trial to examine the effectiveness of generic antimicrobial agents in early dementia.

Alzheimer’s and Infection

In examining the correlation between intracellular infection and Alzheimer’s, it is important to note that the research below pertains largely to Late-Onset Alzheimer’s Disease (LOAD), not the condition linked to APOE ε4 allele on human chromosome 19 (Familial Alzheimer’s Disease).  The etiology of LOAD does not appear to necessarily arise from the expression of that gene.  Evidence provided below indicates that there is ample proof to suggest a correlation between Chlamydia pneumoniae (the predominant intracellular pathogen identified) and the onset of Dementia and Alzheimer’s in a significant number of patients.  Several clues point to an infection.  One is the olfactory pathway which C. pneumoniae uses to enter the CNS.  Damage to olfactory bulbs is present early in the disease as elucidated by Dr. Balin, his colleagues, and other researchers.   C. pneumoniae genetic material was found in these bulbs in post-autopsy LOAD samples.  To quote Dr. Balin “These findings bring into question how specific infection(s), inflammation, and/or damage of the olfactory bulbs could lead to damage in deeper cortical and limbic structures, thereby resulting in symptoms of LOAD”(JAD, 2008)“.  The features of Dementia and Alzheimer’s which include olfactory dysfunction and mitochondrial damage/dysfunction also fit with the infection hypothesis and provide another clue.  Chlamydia pneumoniae steals ATP from mitochondria and has been proven to induce a state of inflammation and elicit mitochondrial damage.

Diagnostic Advancement

Loss or impairment of olfaction (sense of smell) has been identified as an early indicator of Alzheimer’s Disease.  This presents a low-cost, non-invasive, easily administered testing option to help identify patients early in the disease process.  This in conjunction with a traditional MMSE (Mini Mental-State examination) could identify patients that are candidates for antimicrobial therapy before extensive damage and the widespread release of Heat Shock Protein (HSP) 60 and the recruitment of inflammatory cytokines.

Research Studies

A Randomized, Controlled Trial of Doxycycline and Rifampin for Patients with Alzheimer’s Disease
Randomized, triple-blind, controlled trial.  n = 101. JAGS, 2004.
Methods- daily doses of doxycycline 200 mg and rifampin 300 mg for 3 months given to patients with probable AD and mild to moderate dementia.
Results-  There was significantly less decline in the SADAScog score at 6 months in the antibiotic group than in the placebo group, ( – 2.75 points, 95% confidence interval (CI) -5.28 to -0.22, P = .034). At 12 months, the difference between groups in the SADAScog was 4.31 points (95% CI =  -9.17– -0.56, P = .079). The antibiotic group showed significantly less dysfunctional behavior at 3 months. There was no significant difference in adverse events between groups (P = .34). There were no differences in Chlamydia pneumoniae detection using polymerase chain reaction or antibodies (immunoglobulin (Ig)G or IgA) between groups.
Conclusion-  Therapy with doxycycline and rifampin may have a therapeutic role in patients with mild to moderate AD.
Observation- A trend across C. pneumoniae disease-state studies is the variability in detection of organisms using PCR and antibody tests.  This has been attributed to laboratory technique variability, and traits of Chlamydia itself which has been shown to produce unreliable IgG and IgM levels from patient to patient both in the presence and absence of antimicrobial therapy.  Rather than suggesting an absence of the organism, it further elucidates the need for more reliable testing methods and substantiates empiric treatment until such testing becomes available. To quote (Gerard et al. 2006) “Data from this trial did not show that attenuation of decline in psychometric parameters resulted from the effects of antibiotics, but the analyses for change in infection may have been less than fully adequate, because the sampling of antibodies and organism in blood is not always indicative of what has occurred in tissues”.

Immunohistological detection of Chlamydia pneumoniae in the Alzheimer’s disease brain
Post-mortem analysis.  n = 5 AD brains, and 5 normal control brains.  BMC Neuroscience 2010.
Methods- immunohistochemistry was used with a battery of commercially available anti-C. pneumoniae antibodies to determine whether C. pneumoniae was present in areas typically associated with AD neuropathology.
Results– Immunoreactivity for C. pneumoniae antigens was observed both intracellularly in neurons, neuroglia, endothelial cells, and peri-endothelial cells, and extracellularly in the frontal and temporal cortices of the AD brain with multiple C. pneumoniae-specific antibodies. This immunoreactivity was seen in regions of amyloid deposition as revealed by immunolabeling with two different anti-beta amyloid antibodies. Thioflavin S staining, overlaid with C. pneumoniae immunolabeling, demonstrated no direct co-localization of the organism and amyloid plaques. Further, the specificity of C. pneumoniae labeling of AD brain sections was demonstrated using C. pneumoniae antibodies pre-absorbed against amyloid b 1-40 and 1-42 peptides.
Conclusion- Anti-C. pneumoniae antibodies, obtained commercially, identified both typical intracellular and atypical extracellular C. pneumoniae antigens in frontal and temporal cortices of the AD brain. C. pneumoniae, amyloid deposits, and neurofibrillary tangles were present in the same regions of the brain in apposition to one another. Although additional studies are required to conclusively characterize the nature of Chlamydial immunoreactivity in the AD brain, these results further implicate C. pneumoniae infection with the pathogenesis of Alzheimer’s disease.

Chlamydophila (Chlamydia) pneumoniae in the Alzheimer’s brain.
Post-mortem analysis of brain-tissue samples.  n =  25 with late-onset AD and 27 non-AD control individuals. Federation of European Microbiological Societies, 2006.
Methods- Postmortem brain-tissue samples from the hippocampus, temporal cortex, frontal cortex, parietal cortex, and/or occipital lobe were obtained under an approved International Review Board (IRB).  PCR, Iummunohistochemistry, C. pneumonaie culture, and in situ hybridization were employed.
Results- 20/27 AD patients, but only 3/27 controls, were PCR-positive in multiple assays targetting the Cpn1046 and Cpn0695 genes. Culture of the organism from brain-tissue homogenate from one AD patient, and assessment of various chlamydial transcripts in RNA preparations from several patients, demonstrated that the organisms were viable and metabolically active in those samples. Immunohistochemical analyses showed that astrocytes, microglia, and neurons all served as host cells for C. pneumoniae in the AD brain, and that infected cells were found in close proximity to both neuritic senile plaques and neurofibrillary tangles in the AD brain. These observations confirm and significantly extend our earlier study suggesting that this unusual pathogen may play a role in the neuropathogenesis characteristic of AD.
Conclusions- We confirmed that astrocytes and microglia are common host cell types for the organism in the AD brain, and importantly we demonstrated that c. 20% of neurons are infected as well. Finally, we confirmed that C. pneumoniae-infected cells in the AD brain are found in close association with NFT, and we provided evidence that infected cells also are commonly found in areas of NSP. While none of these observations demonstrates a causal relationship between CNS infection with C. pneumoniae and the neuropathogenesis characteristic of AD, they are consistent with such a relationship.

The load of Chlamydia pneumoniae in the Alzheimer’s brain varies with APOE genotype
Microbial Pathogenesis, 2005.
Studies from this laboratory have indicated that the intracellular eubacterial respiratory pathogen Chlamydophila (Chlamydia) pneumoniae is commonly found in brain regions displaying characteristic neuropathology in patients with late-onset Alzheimer’s disease (AD) but not in congruent samples from non-AD control individuals. In later work, we provided evidence suggesting that some relationship exists between the APOE epsilon4 gene product and the pathobiology of this organism.
Methods- In the present report, in situ hybridization analyses indicated that the number of C. pneumoniae-infected cells in affected brain regions of epsilon4-bearing AD patients was higher overall than that in congruent brain regions from AD patients lacking that allele.
Results- Quantitative real-time PCR analyses of AD brain tissue samples demonstrated that actual bacterial burden in those samples varied over several orders of magnitude, but that samples from epsilon4-bearing patients did have significantly higher bacterial loads than did congruent samples from patients without the allele (ANOVA, p<0.05).
Conclusion- These results may explain in part the observations that epsilon4-bearing individuals have a higher risk of developing AD, and that such patients progress more rapidly to cognitive dysfunction than do individuals lacking this allele.

Identification and localization of Chlamydia pneumoniae in the Alzheimer’s brain
Post-mortem brain tissue analysis, n= 38, Medical Microbiology and Immunology, 1998.
Methods- Nucleic acids prepared from those samples were screened by polymerase chain reaction (PCR) assay for DNA sequences from the bacterium.
Results- Analyses showed that brain areas with typical AD-related neuropathology were positive for the organism in 17/19 AD patients. Similar analyses of identical brain areas of 18/19 control patients were PCR-negative. Electron- and immunoelectron-microscopic studies of tissues from affected AD brain regions identified chlamydial elementary and reticulate bodies, but similar examinations of non-AD brains were negative for the bacterium. Culture studies of a subset of affected AD brain tissues for C. pneumoniae were strongly positive, while identically performed analyses of non-AD brain tissues were negative. Reverse transcription (RT)-PCR assays using RNA from affected areas of AD brains confirmed that transcripts from two important C. pneumoniae genes were present in those samples but not in controls. Immunohistochemical examination of AD brains, but not those of controls, identified C. pneumoniae within pericytes, microglia, and astroglia. Further immunolabelling studies confirmed the organisms’ intracellular presence primarily in areas of neuropathology in the AD brain.
Conclusion- Thus, C. pneumoniae is present, viable, and transcriptionally active in areas of neuropathology in the AD brain, possibly suggesting that infection with the organism is a risk factor for late-onset AD.

Inhibition of apoptosis in neuronal cells infected with Chlamydophila (Chlamydia) pneumoniae
Post-mortem brain tissue analysis, BMC Neuroscience, 2008.
Abstract- Chlamydophila (Chlamydia) pneumoniae is an intracellular bacterium that has been identified within cells in areas of neuropathology found in Alzheimer disease (AD), including endothelia, glia, and neurons. Depending on the cell type of the host, infection by C. pneumoniae has been shown to influence apoptotic pathways in both pro- and anti-apoptotic fashions. We have hypothesized that persistent chlamydial infection of neurons may be an important mediator of the characteristic neuropathology observed in AD brains. Chronic and/or persistent infection of neuronal cells with C. pneumoniae in the AD brain may affect apoptosis in cells containing chlamydial inclusions.
Methods-  SK-N-MC neuroblastoma cells were infected with the respiratory strain of C. pneumoniae, AR39 at an MOI of 1. Following infection, the cells were either untreated or treated with staurosporine and then examined for apoptosis by labeling for nuclear fragmentation, caspase activity, and membrane inversion as indicated by annexin V staining. C. pneumoniae infection was maintained through 10 days post-infection.
Results- At 3 and 10 days post-infection, the infected cell cultures appeared to inhibit or were resistant to the apoptotic process when induced by staurosporine. This inhibition was demonstrated quantitatively by nuclear profile counts and caspase 3/7 activity measurements.
Conclusion- These data suggest that C. pneumoniae can sustain a chronic infection in neuronal cells by interfering with apoptosis, which may contribute to chronic inflammation in the AD brain.

Ultrastructural Analysis of Chlamydia Pneumoniae in the Alzheimer’s Brain
Post-mortem brain tissue analysis, n= 4, Pathogenesis, 1999.
Abstract- We recently reported identification of the bacterium Chlamydia pneumoniae in affected brain regions of patients with Alzheimer’s disease (AD) (Balin et al., 1998). In this report, we extend those initial observations to demonstrate that, in addition to the frequently described, standard morphological forms of the organism, pleiomorphic forms are also present in the AD brain.
Methods- All AD and control brain tissues examined were verified to be PCR-positive and negative, respectively, for the organism. DNA sequence determination of PCR products so derived from total DNA of infected AD brains, as well as from total DNA of cell lines infected with the organism following isolation from these same patient samples, confirmed the presence of organism in relevant samples.
Results-  morphologic forms of C. pneumoniae were identified in PCR-positive tissues and these were characterized based on membrane structure, core density, size, and immunolabeling profiles. Structures identified include the typical pear-shaped elementary body, as well as larger, spherical and oblong reticulate bodies. Intact C. pneumoniae were found both intracellularly and extracellularly in the sampled autopsy brains. Intracellular organisms were located principally within microglia, astroglia, and presumptive pericytes.
Conclusion- C. pneumoniae found in cells indigenous to the AD brain do not conform universally to the classical morphology observed in other infected cell types. This pleiomorphism may reflect an adaptive response and/or persistent state of infection for these organisms in Alzheimer’s Disease.

Initial characterization of Chlamydophila (Chlamydia) pneumoniae cultured from the late-onset Alzheimer brain
Post-mortem tissue analysis, n= 2 LOAD brains, Int. Journal of Med. Microbiology, 2009.
Method- Here we report culture of the organism from two AD brain samples, each of which originated from a different geographic region of North America.
Results- Culturable organisms were detectable after one and two passages in HEp-2 cells, respectively, for the two samples. Both isolates, designated Tor-1 and Phi-1, were demonstrated to be authentic C. pneumoniae using PCR assays targeting the C. pneumoniae-specific genes Cpn0695, Cpn1046, and tyrP. Assessment of inclusion morphology and quantitation of infectious yields in epithelial (HEp-2), astrocytic (U-87 MG), and microglial (CHME-5) cell lines demonstrated an active, rather than a persistent, growth phenotype for both isolates in all host cell types. Sequencing of the omp1 gene from each isolate, and directly from DNA prepared from several additional AD brain tissue samples PCR-positive for C. pneumoniae, revealed genetically diverse chlamydial populations. Both brain isolates carry several copies of the tyrP gene, a triple copy in Tor-1, and predominantly a triple copy in Phi-1 with a minor population component having a double copy.
Conclusion- This observation indicated that the brain isolates are more closely related to respiratory than to vascular/atheroma strains of C. pneumoniae

Chlamydia pneumoniae infection of monocytes in vitro stimulates innate and adaptive immune responses relevant to those in Alzheimer’s disease
Journal of Neuroinflammation, 2014.
Methods- Gene transcription was analyzed by RT-PCR using an innate and adaptive immunity microarray with 84 genes organized into 5 functional categories: inflammatory response, host defense against bacteria, antibacterial humoral response, septic shock, and cytokines, chemokines and their receptors. Statistical analysis of the results was performed using the Student’s t-test. P-values ≤ 0.05 were considered to be significant. ELISA was performed on supernatants from uninfected and Cpn-infected THP1 monocytes followed by statistical analysis with ANOVA.
Results- When Cpn-infected THP1 human monocytes were compared to control uninfected monocytes at 48 hours post-infection, 17 genes were found to have a significant 4-fold or greater expression, and no gene expression was found to be down-regulated. Furthermore, cytokine secretion (IL-1β, IL-6, IL-8) appears to be maintained for an extended period of infection.
Conclusions- Utilizing RT-PCR and ELISA techniques, our data demonstrate that Cpn infection of THP1 human monocytes promotes an innate immune response and suggests a potential role in the initiation of inflammation in sporadic/late-onset Alzheimer’s disease.

Evaluation of CSF-Chlamydia pneumoniae, CSF-tau, and CSF-Abeta42 in Alzheimer’s disease and vascular dementia
Prospective analysis of CSF in LOAD, Vascular Dementia versus a control group,
n= 125, Journal of Neurology, 2007.
Methods- 1mL of CSF was collected, the DNA was extracted using the chelex method according to the Walsh protocol. Tau was measured using a commercial enzyme immunoassay.
Results- In this study, the appearance of Chlamydia pneumoniae (CpN) in the cerebrospinal fluid (CSF) of 57 AD and 21 VD patients and in 47 controls (CG), as well as the influence of CpN on the levels of tau protein and Ab42, were investigated. The frequency of CpN occurrence in the AD patient group (43.9%) was significantly higher (p < 0.001) than in the control group (10.6%). In the case of VD patients, 9.5% of this group was positive for CpN The presence of CpN DNA in the CSF of patients with AD significantly increases the occurrence of this disease (odds ratio = 7.21). Cerebrospinal fluid Ab42 levels were significantly lower in patients with AD than in the CG (p < 0.001). Cerebrospinal tau protein was significantly higher in AD vs. CG (p = 0.007). However, no relationships between the presence of the bacterium in CSF and the level of either tau or Ab42 protein were observed.
Conclusion- testing for the presence of CpN in CSF, along with the tau and Ab42 markers, may be used in the clinical diagnosis of AD.

Brian J. Balin, Ph.D. and Nikki Schultek, Principal & Founder of Intracell Research Group

In vitro study, using immunofluorescent microscopy, molecular, and biochemical approaches.  BMC Neuroscience, 2019.
Background- “Epidemiologic studies strongly suggest that the pathophysiology of late-onset Alzheimer disease (AD) versus early-onset AD has environmental rather than genetic causes, thus revealing potentially novel therapeutic targets to limit disease progression. Several studies supporting the “pathogen hypothesis” of AD demonstrate a strong association between pathogens and the production of β-amyloid, the pathologic hallmark of AD. Although the mechanism of pathogen-induced neurodegeneration of AD remains unclear, astrocytes, a key player of the CNS innate immune response and producer/metabolizer of β-amyloid, have been implicated. We hypothesized that Chlamydia pneumoniae infection of human astrocytes alters the expression of the amyloid precursor protein (APP)-processing secretases, ADAM10, BACE1, and PSEN1, to promote β-amyloid formation. Utilizing immunofluorescent microscopy, molecular, and biochemical approaches, these studies explore the role of an intracellular respiratory pathogen, Chlamydia pneumoniae, as an environmental trigger for AD pathology. “
Methods- “Human astrocytoma cells in vitro were infected with Chlamydia pneumoniae over the course of 6–72 h. The gene and protein expression, as well as the enzymatic activity of non-amyloidogenic (ADAM10), and pro-amyloidogenic (BACE1 and PSEN1) secretases were qualitatively and quantitatively assessed. In addition, the formation of toxic amyloid products as an outcome of pro-amyloidogenic APP processing was evaluated through various modalities.”
Results- “Chlamydia pneumoniae infection of human astrocytoma cells promoted the transcriptional upregulation of numerous genes implicated in host neuroinflammation, lipid homeostasis, microtubule function, and APP processing. Relative to that of uninfected astrocytes, BACE1 and PSEN1 protein levels were enhanced by nearly twofold at 48–72 h post-Chlamydia pneumoniae infection. The processing of APP in Chlamydia pneumoniae-infected astrocytes favors the pro-amyloidogenic pathway, as demonstrated by an increase in enzymatic activity of BACE1, while that of ADAM10 was decreased. Fluorescence intensity of β-amyloid and ELISA-quantified levels of soluble-APP by products revealed temporally similar increases, confirming a BACE1/PSEN1-mediated processing of APP.”
Conclusions- “Our findings suggest that Chlamydia pneumoniae infection of human astrocytes promotes the pro-amyloidogenic pathway of APP processing through the upregulation of expression and activity of β-secretase, upregulated expression of γ-secretase, and decreased activity of α-secretase. These effects of astrocyte infection provide evidence for a direct link between Chlamydia pneumoniae and AD pathology.”

Olfactory Loss in Alzheimer’s

Odor identification as a biomarker of preclinical AD in older adults at risk
Cohort analysis, n= 274, 101 CSF donations.  Neurology, 2017.
Methods- OI measured using the University of Pennsylvania Smell Identification Test and cognitive performance using the Repeatable Battery for Assessment of Neuropsychological Status.  Assessment of AD pathology with the biomarkers total tau (t-tau), phospho-tau (P181-tau), and their ratios with β-amyloid (Aβ1-42). Adjusted analyses considered age, cognition, APOE ε4 status, education, and sex as covariates.
Results- Reduced OI was associated with lower cognitive score and older age, as well as increased ratios of CSF t-tau and P181-tau to Aβ1-42 (all p < 0.02).
Conclusion- These findings from healthy high-risk older individuals suggest that OI reflects the degree of preclinical AD pathology, while its relationships with age and cognition result from the association of these latter variables with such pathology. Diminished OI may be a practical and affordable biomarker of AD pathology.

Early olfactory involvement in Alzheimer’s disease
Autopsy cases, n= 110.  The Canadian Journal of Neurological Sciences, 2003.
Methods- cortical areas and the olfactory bulb and tract were analyzed using histo- and immunohistochemical techniques.  Based on a semiquantitative analysis of cortical senile plaques, neurofibrillary tangles and curly fibers, the 110 cases were divided into four groups: 19 cases with severe (definite AD), 14 cases with moderate, 58 cases with discrete and 19 control cases without AD-type cortical changes.
Results- The number of cases with olfactory involvement was very high, more than 84% in the three groups with cortical AD-type lesions. Degenerative olfactory changes were present in all 19 definite AD cases, and in two of the 19 controls. The statistical analysis showed a significant association between the peripheral olfactory and cortical degenerative changes with respect to their frequency and severity (P < 0.001). Neurofibrillary tangles and neuropil threads appear in the olfactory system as early as in entorhinal cortex.

Olfactory centres in Alzheimer’s disease: olfactory bulb is involved in early Braak’s stages
post-mortem tissue and case analysis, n= 30 (15 AD cases compared with 15 non-AD cases), Neuroreport, 2001.
Methods- Cases asssessed by psychiatrist using NINCDS-ADRDA criteria, CERAD and Braak and Braak staging.  Brain tissue assessed post-mortem using immunohistochemistry.
Results- statistically significant difference in brain weight between AD and controls suggesting atrophy in AD (p=.002), all but one patient had neurofibrillary tangles, the anterior olfactory nucleus and olfactory bulbs were damaged in AD relative to controls.
Conclusion- Olfactory bulbs damaged earlier in AD than entorhinal cortex.  This supports the belief that olfactory testing is a preclinical marker for AD.

A Brief Olfactory Test for Alzheimer’s Disease
Retrospective, case-control study.  n = 94.  Journal of Neurological Sciences, 2014.
Design- Participants with probable AD (N=18), mild cognitive impairment (MCI, N=24), other causes of dementia (OD, N=26) and matched controls (OC, N=26) were tested, with closed eyes, for their ability to detect an odor, one nostril at a time. A container of 14g of peanut butter was opened, held medially at the bottom of a 30 cm ruler, and moved up 1cm at a time during the participants’ exhale. Upon odor detection, the distance between the subject’s nostril and container was measured.
Results- The mean odor detection distance of AD patients’ left nostril (5.1 cm), and not their right (17.4 cm), was significantly less (F(3,90) = 22.28, p < 0.0001) than the other groups. The mean, standard error, and 95% Confidence Interval of the L R nostril odor detection difference (cm) for AD was −12.4 ±0.5, (−15.0, −9.8); for MCI was −1.9 ±1.2, (−4.2,0.4); for OD was 4.8 ±1.0, (2.6,6.9); and for OC was 0.0 ±1.4 (−2.2,2.1).
Conclusion- This non-invasive and inexpensive left-right nostril odor detection test appears to be a sensitive and specific test for probable AD.

Pathologic changes in olfactory neurons in Alzheimer’s disease
Annals New York Academy of Sciences, 1991.
Abstract- “We speculated that the peripheral olfactory neurons in the nose, which are readily available for biopsy, might be affected in AD and might be a valuable source of tissue for examination and studies of the mechanism and progression of the disease process. The importance of identifying a peripheral site that is affected in AD is clear because our ability to study the development of degenerative events and to diagnose AD is severely restricted by the inaccessibility of the affected brain tissue, which lies within the cranium and is not easily obtained for the purposes of biopsy.”

SMELL-S and SMELL-R: Olfactory tests not influenced by odor-specific insensitivity or prior olfactory experience
PNAS, 2017.
Abstract- “Smell dysfunction is a common and underdiagnosed medical condition that can have serious consequences. It is also an early biomarker of neurodegenerative diseases, including Alzheimer’s disease, where olfactory deficits precede detectable memory loss. Clinical tests that evaluate the sense of smell face two major challenges. First, human sensitivity to individual odorants varies significantly, so test results may be unreliable in people with low sensitivity to a test odorant but an otherwise normal sense of smell.”

Olfactory Identification Deficits, Cognitive Decline, and Dementia in Older Adults
American Journal of Geriatric Psychiatry, 2016.
Abstract- “Several recently developed biomarkers of Alzheimer disease (AD) are invasive, expensive, and difficult to obtain in most clinical settings. Olfactory identification test performance represents a noninvasive, inexpensive biomarker of AD that may have predictive accuracy comparable with neuroimaging measures and biomarkers assessed in cerebrospinal fluid. Neurofibrillary tangles in the olfactory bulb are among the earliest pathologic features of AD and are also seen in the projection pathways from the olfactory bulb to secondary olfactory brain regions, including the piriform and medial temporal cortex, orbitofrontal cortex, and other limbic regions. Odor identification impairment characterizes AD and predicts the clinical transition from mild cognitive impairment to AD in both clinical and community samples.”

Evaluation of olfactory dysfunction in neurodegenerative diseases
Review Article, Journal of the Neurological Sciences, 2012.
Parkinson’s disease, Alzheimer’s disease, multiple sclerosis, Huntington’s disease and motor neuron disease have all been associated with olfactory dysfunction.  The review discusses the use of olfactory event-related potentials (OERPs) and functional magnetic resonance imaging (fMRI) measurements as markers to assist in earlier diagnosis of the aforementioned diseases.

Mouse Models

Antibiotic alters inflammation in the mouse brain during persistent Chlamydia pneumoniae infection
10th International Conference on Alzheimer’s Disease and Related Disorders, 2006.
Methods- Mouse model assessing whether moxifloxacin hydrochloride had an effect on the inflammatory process and deposition of amyloid in the mouse brain during persistent Chlamydia pneumoniae infection.
Result- with early antibiotic treatment, astrocytic activation was more prominent at 3 months.
Conclusion- early antibiotic treatment may modulate the cellular inflammatory processes following infection and, ultimately, the extent of amyloid deposition in the mouse brain.

Chlamydia pneumoniae induces Alzheimer-like amyloid plaques in brains of BALB/c mice
Neurobiology of Aging, 2004.
Abstract- “Amyloid deposits resembling plaques found in Alzheimer’s disease (AD) brains were formed in the brains of non-transgenic BALB/c mice following intranasal infection with Chlamydia pneumoniae. The mice were infected at 3 months of age with C. pneumoniae isolated from an AD brain. Infection was confirmed by light and electron microscopy in olfactory tissues of the mice. C. pneumoniae was still evident in these tissues 3 months after the initial infection indicating that a persistent infection had been established. Amyloid beta (A ) 1–42 immunoreactive deposits were identified in the brains of infected BALB/c mice up to 3 months post-infection with the density, size, and number of deposits increasing as the infection progressed. A subset of deposits exhibited thioflavin-s labeling. Intracellular A 1–42 labeling was observed in neuronal cells. Experimental induction of amyloid deposition in brains of non-transgenic BALB/c mice following infection with C. pneumoniae may be a useful model for furthering our understanding of mechanisms, linked to infection, involved in the initiation of the pathogenesis of sporadic AD.”

Age Alterations in Extent and Severity of Experimental Intranasal Infection with Chlamydophila pneumoniae in BALB/c Mice
Infection and Immunity, 2005.
Assessed whether aging is coupled with increased burden of infection in BALB/c mice after intranasal infection by C. pneumoniae.
Methods- Six- and twenty-month-old BALB/c mice were infected intranasally with 5 104 inclusion forming units (IFU) or 5 105 IFU of C. pneumoniae. Lung, brain, and heart tissue were analyzed for infectious C. pneumoniae and for Chlamydophila antigen by immunohistochemistry.
Results- At both doses, aging was associated with a decreased proportion of animals that cleared infection from the lung and greater burden of infectious organism within the lung. We observed dose-dependent spread to the heart/ascending aorta in animals infected with C. pneumoniae. In mice given 5 104 IFU, spread to the heart by day 14 was only observed in old mice. By day 28, all animals inoculated with 5 104 IFU showed evidence of spread to the heart, although higher C. pneumoniae titers were observed in the hearts from old mice. In mice inoculated with 5 105 IFU, spread of C. pneumoniae to the heart was evident by day 14, with no discernible age effect. C. pneumoniae was also recovered from the central nervous system (brain and olfactory bulb) of all mice by day 28 postinfection, with higher C. pneumoniae titers in old animals than in young animals. Conclusion- Our results suggest that infection with C. pneumoniae may be more severe in old animals.

Review Articles

Chlamydia pneumoniae: An Etiologic Agent for Late-Onset Dementia
Frontiers in Aging Neuroscience, 2018.
Dr. Balin and colleagues review the work surrounding the correlation between CpN infection and the development of dementia.  To quote the authors:

“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. “

Herpes viruses and Alzheimer’s disease: new evidence in the debate
Lancet Neurology, 2018.
Dr. Balin and Dr. Hudson were asked by Lancet Neurology to draft a response to the recent work by Dr. Tanzi and colleagues correlating LOAD to HSV-1 infection.  Here is the most powerful message from the article.

“With the focus having been on amyloid-β and tau for the past 30 years and more, we contend that answers to the questions of Alzheimer’s disease, and possibly other neurodegenerative conditions as well, does not reside solely in the pathology, but rather in the route to that pathology, for which infectious agents provide biologically and pathologically relevant solutions.”

Chlamydia Pneuominae as an Etiologic Agent for Late-Onset Alzheimer’s Disease

IOS Press, 2017.
This review focuses specifically on infection with Chlamydophila (Chlamydia) pneumoniae in LOAD and how this infection may function as a “trigger or initiator” in the pathogenesis of this disease.

Microbes and Alzheimer’s Disease
Journal of Alzheimer’s Disease, 2016.
Abstract- “We are researchers and clinicians working on Alzheimer’s disease(AD) or related topics, and we write to express our concern that one particular aspect of the disease has been neglected, even though treatment based on it might slow or arrest AD progression. We refer to the many studies, mainly on humans, implicating specific microbes in the elderly brain, notably herpes simplex virus type1(HSV1), Chlamydia pneumoniae, and several types of spirochaete, in the etiology ofAD.  Fungal infection of AD brain[5,6]has also been described, as well as abnormal microbiota in AD patient blood [7].  The first observations of HSV1 in AD brain were reported almost three decades ago[8].  The ever-increasing number of these studies (now about 100 on HSV1 alone) warrants re-evaluation of the infection and AD concept.”

Herpes Simplex Virus Type 1 and Other Pathogens are Key Causative Factors
in Sporadic Alzheimer’s Disease
Journal of Alzheimer’s Disease, 2015
This review explores the relationship between pathogens, including Herpes simplex virus type 1 (HSV-1), Cytomegalovirus, and other Herpesviridae, Chlamydophila pneumoniae, spirochetes, Helicobacter pylori, and various periodontal pathogens and sporadic Alzheimer’s Disease.  It examines the correlation of infection with the release of pro- inflammatory cytokines that may cross the blood-brain barrier and promote neurodegeneration.

Infectious Agents and Neurodegeneration
Molecular Neurobiology, December 2012.
Abstract- “A growing body of epidemiologic and experimental data point to chronic bacterial and viral infections as possible risk factors for neurodegenerative diseases, including Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis. Infections of the central nervous system, especially those characterized by a chronic progressive course, may produce multiple damage in infected and neighboring cells. The activation of inflammatory processes and host immune responses cause chronic damage resulting in alterations of neuronal function and viability, but different pathogens can also directly trigger neurotoxic pathways. Indeed, viral and microbial agents have been reported to produce molecular hallmarks of neurodegeneration, such as the production and deposit of misfolded protein aggregates, oxidative stress, deficient autophagic processes, synaptopathies and neuronal death. These effects may act in synergy with other recognized risk factors, such as aging, concomitant metabolic diseases and the host’s specific genetic signature. This review will focus on the contribution given to neurodegeneration by herpes simplex type-1, human immunodeficiency and influenza viruses, and by Chlamydia pneumoniae.”

Historic evidence to support a causal relationship between spirochetal infections and Alzheimer’s disease
Frontiers In Aging Neuroscience, 2015.
Abstract- “Following previous observations a statistically significant association between various types of spirochetes and Alzheimer’s disease (AD) fulfilled Hill’s criteria in favor of a causal relationship. If spirochetal infections can indeed cause AD, the pathological and biological hallmarks of AD should also occur in syphilitic dementia.  Historic observations and illustrations published in the first half of the 20th Century indeed confirm that the pathological hallmarks, which define AD, are also present in syphilitic dementia. Cortical spirochetal colonies are made up by innumerable tightly spiraled Treponema pallidum spirochetes, which are morphologically indistinguishable from senile plaques, using conventional light microscopy. Local brain amyloidosis also occurs in general paresis and, as in AD, corresponds to amyloid beta. These historic observations enable us to conclude that chronic spirochetal infections can cause dementia and reproduce the defining hallmarks of AD.”

Chlamydophila pneumoniae and the etiology of late-onset Alzheimer’s disease.
Journal of Alzheimers Disease, 2008.
Abstract- “Sporadic, late-onset Alzheimer’s disease (LOAD) is a non-familial, progressive neurodegenerative disease that is now the most common and severe form of dementia in the elderly. That dementia is a direct result of neuronal damage and loss associated with accumulations of abnormal protein deposits in the brain. Great strides have been made in the past 20 years with regard to understanding the pathological entities that arise in the AD brain, both for familial AD (∼5% of all cases) and LOAD (∼95% of all cases). The neuropathology observed includes: neuritic senile plaques (NSPs), neurofibrillary tangles (NFTs), neuropil threads (NPs), and often deposits of cerebrovascular amyloid. Genetic, biochemical, and immunological analyses have provided a relatively detailed knowledge of these entities, but our understanding of the “trigger” events leading to the many cascades resulting in this pathology and neurodegeneration is still quite limited. For this reason, the etiology of AD, in particular LOAD, has remained elusive. However, a number of recent and ongoing studies have implicated infection in the etiology and pathogenesis of LOAD. This review focuses specifically on infection with Chlamydophila (Chlamydia) pneumoniae in LOAD and how this infection may function as a “trigger or initiator” in the pathogenesis of this disease.”

Infiltration of the brain by pathogens causes Alzheimer’s disease.
Neurobiology of Aging, 2004.
Abstract- “Despite very numerous studies on Alzheimer’s disease (AD), especially on amyloid plaques and neurofibrillary tangles, little information has been obtained thus on the causes of the disease. Evidence is described here that implicates firstly herpes simplex virus type 1 (HSV1) as a strong risk factor when it is present in brain of carriers of the type 4 allele of the gene for apolipoprotein E (APOE-4). Indirect support comes from studies indicating the role of APOE in several diverse diseases of known pathogen cause. A second putative risk factor is the bacterium, Chlamydia pneumoniae. This pathogen has been identified and localized in AD brain. Current studies aimed at “proof of principle” address the entry of the organism into the CNS, the neuroinflammatory response to the organism, and the role that the organism plays in triggering AD pathology. An infection-based animal model demonstrates that following intranasal inoculation of BALB/c mice with C. pneumoniae, amyloid plaques/deposits consistent with those observed in the AD brain develop, thus implicating this infection in the etiology of AD.”

Challenges and directions for the pathogen hypothesis of Alzheimer’s disease.
Neurobiology of Aging, 2004.
Abstract- “This paper critically reviews the possibility that infiltration of the brain by pathogens (e.g. Herpes simplex virus type 1 (HSV1) or Chlamydophila pneumoniae (Cp)) acts as a trigger or co-factor for Alzheimer’s disease (AD). The evidence currently available is limited and in some cases inconsistent, but it does justify the need for more vigorous investigation of this hypothesis. An issue of particular concern is the paucity of experimental evidence showing that pathogens can elicit the neuropathological changes and cognitive deficits that characterise AD. Other weaknesses include a failure to obtain independent confirmation of Cp in AD brains, and a lack of evidence for HSV1 proteins or intact virions in AD brain tissue. Future avenues of investigation that might prove fruitful include epidemiological investigations of the incidence of AD in individuals who are either immunosuppressed or have received chronic antiviral or antibiotic therapy. There is also a need to consider systemic infections as potential contributors to the pathogenesis of AD.”

Alzheimer’s disease and infection: do infectious agents contribute to progression of Alzheimer’s disease?
Alzheimers and Dementia, 2009.
Abstract- “Infection with several important pathogens could constitute risk factors for cognitive impairment, dementia, and Alzheimer’s disease (AD) in particular. This review summarizes the data related to infectious agents that appear to have a relationship with AD. Infections with herpes simplex virus type 1, picornavirus, Borna disease virus, Chlamydia pneumoniaeHelicobacter pylori, and spirochete were reported to contribute to the pathophysiology of AD or to cognitive changes. Based on these reports, it may be hypothesized that central nervous system or systemic infections may contribute to the pathogenesis or pathophysiology of AD, and chronic infection with several pathogens should be considered a risk factor for sporadic AD. If this hypothesis holds true, early intervention against infection may delay or even prevent the future development of AD.”

What is the relationship between Chlamydia pneumoniae and Late-onset Alzheimer’s Disease?
Laboratory Medicine, 2001.
Abstract- “Beginning with the demonstration of Helicobacter pylori as an etiologic agent for ulcers, the notion that microbial pathogens can play an initiating and/or exacerbatory role in chronic disease has gained increasing credence. Indeed, studies have suggested associations between Chlamydia trachomatis and cervical cancer, Nocardia asteroides and Parkinson’s disease, and Chlamydia pneumoniae and atherosclerosis, among many others. While most such associations are controversial, these lines of research remain under active investigation. In this article, evidence for and against the association between late-onset Alzheimer’s disease (AD) and persistent infection with C. pneumoniae will be reviewed.”

Are the Infectious Roots of Alzheimers Buried Deep in the Past?
Journal of Molecular Pathological Epidemiology, 2017.
 “Recent literature shows a controversial new push to tie microorganisms to Alzheimer’s disease (AD). Study after study, in which scientists have injected human Alzheimer-diseased brain tissue into mice and other laboratory animals that later developed the disease have left little doubt that Alzheimer’s disease (AD) arises from an infectious process. By 2013 Mawanda and Wallace’s “Can Infections Cause Alzheimer’s Disease” struck down some of the commonly entertained pathogens for AD such as herpes simplex virus type 1, Chlamydia pneumoniae, and several types of spirochetes. Instead, they pointed to two prime suspects for Alzheimer’s amyloid-beta deposition: “especially chronic infections like tuberculosis and leprosy.” To be sure, it was German neuropathologist Oskar Fischer of the Prague school of Neuropathology, Alzheimer’s great rival, who was the first to suggest that infection might be causative for Alzheimer’s. Fischer’s credentials: he was the co-discoverer of Alzheimer’s disease. His suspected germ was the Streptothrix, today classified as Actinomycetes, a rare central nervous system pathogen which at the time was so constantly and consistently mistaken for tuberculosis that Choppen- Jones suggested that TB be called tuberculomycosis. And Just ten years before Oskar Fischer found Actinomycosis-like forms in Alzheimer’s cerebral plaque, Babèş and immunologist Levaditi reported in “On the Actinomycotic Shape of the Tuberculous Bacilli” that Fischer’s typical Actinomyces-like clusters (Drüsen) with clubs appeared in the tissue of rabbits inoculated with tubercle bacilli beneath the dura mater of their brains. Investigators who supported and subsequently followed up on Fischer’s Alzheimer’s germ are also discussed.”