Chlamydia pneumoniae

Alzheimer’s and Chlamydia

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 (a predominant intracellular bacterial 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.


Research Studies

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.
Bakground-
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.


NEW
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.”

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. “



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.



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.”


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.”