Our previous studies of brain tissues obtained at autopsy have established an association of Chlamydia pneumoniae (Cpn) infection with late‐onset Alzheimer’s disease (AD). Given these findings, we have designed a study using electron microscopy (EM) to determine if various forms of Cpn from geriatric patient’s blood samples could be identified and correlated with cognitive decline associated with AD.Patients, >65 years old, and who participated in this IRB approved study, met well‐defined exclusion criteria prior to being evaluated for cognitive status using the SLUMS and FAST testing assessments. After psychometric testing, blood was drawn from the patients and these samples were prepared for EM and assessed for the presence of Cpn.Our ultrastructural analysis revealed evidence for the presence of Cpn in peripheral blood cells from 12 patients correlated with MCI and AD. Intravacuolar reticulate bodies, elementary bodies, and intermediate bodies characteristic of Cpn were observed in leukocytes from the buffy coat. More specifically, we observed the classical “pear‐shaped” elementary bodies characteristic of Cpn. Immuno‐ EM was performed for positive identification of these various forms of Cpn.Our data indicate that screening for Cpn from blood samples may prove to be a viable biomarker in differentiating cognitive change consistent with stages associated with MCI and AD. Investigating the distinct morphologies of Cpn in human blood samples from the geriatric population by EM, with other frontline diagnostic procedures, may clarify the diagnosis of MCI and late onset AD.Grant Funding Source: Center for Chronic Disorders of Aging

Previous studies from our laboratory of brain tissues obtained at autopsy have established an association of Chlamydia pneumoniae infection with late-onset Alzheimer's disease. Given these findings, we have designed a translational research study to identify biomarkers associated with Chlamydia pneumoniae infection in the blood of geriatric patients. These biomarkers may be useful as a diagnostic screen in the evaluation of patients for cognitive change consistent with mild cognitive impairment and Alzheimer's disease. This translational investigation was approved by the PCOM Institutional Review Board prior to patient recruitment and evaluation. In order to participate, identified patients had to be >65 years old and meet well-defined inclusion criteria. Once informed consent was obtained, these patients were evaluated for cognitive status using the SLUMS and FAST testing assessments. After psychometric testing, blood was drawn from the patients and the buffy coat was isolated and assessed by serology, immunofluorescence microscopy, and electron microscopy to identify biomarkers of infection. All methods revealed evidence for Chlamydia pneumoniae infection in a subgroup of our patient population. Current analysis of 16 patients by serology demonstrated 14/16 positive for at least one immunoglobulin class, IgM, IgA, or IgG. Analysis by immunofluorescence microscopy of peripheral blood revealed 13/16 patients positive for Chlamydia when labeled with a genus-specific anti-Chlamydial antibody. Of these 13 patients, 6 demonstrated a very strong positivity in monocyte populations. Limited infection was observed for the remaining positive 7 patients. For additional confirmatory studies to identify Chlamydia pneumoniae infection, a subpopulation of the 13 patient samples was prepared for electron microscopy. In these samples, elementary bodies, intra-vacuolar reticulate bodies, and intermediate bodies, characteristic of Chlamydia, were observed in leukocytes from the buffy coat. More specifically, we observed the classical “pear-shaped” elementary bodies characteristic of Chlamydia pneumoniae. Our data indicate that biomarkers for Chlamydia pneumoniae are present in blood samples from the geriatric patients who participated in this study. These data suggest that these biomarkers could be useful as a diagnostic screen for the geriatric population who may be at risk for developing cognitive decline.

Our laboratory has been studying the role of infection with Chlamydia pneumoniae in sporadic late-onset Alzheimer disease (LOAD). This infection may be a trigger for the pathology observed in LOAD as a function of initiating changes in gene regulation following entry of the organism into the brain. We have shown that entry and infection in the brain may result from infection of both blood-borne monocytes and olfactory neuroepithelial cells. Our current studies focus on infection of human THP1 monocytes, olfactory neuroepithelial cells, and SKNMC neuronal cells in vitro followed by analysis of infection using immunofluorescence and Alzheimer pathway-specific Real-Time PCR microarrays. The different cell types were infected for 48hrs with the laboratory strain of Chlamydia pneumoniae, AR39, at a multiplicity of infection = 0.5-1. Following infection, cells were analyzed by immunocytochemistry using immunofluorescent tagged antibodies. To determine gene regulation changes, RNA extraction was followed by cDNA first strand synthesis and Real-Time PCR. C. pneumoniae prominently and stably infected all cell types at 48hr post-infection. Numerous large inclusions were labelled using anti-chlamydial monoclonal antibodies. Gene expression was altered dramatically in all three cell types. Of the genes up-regulated, only 6 were in common to all 3 cell types at 48hr pi (ACHE, BACE2, CLU, GSK3b, NCSTN, and PRKI), and of the genes down-regulated, only 3 were in common to all 3 cell types (BDNF, INSR, and CTSG). When comparing one cell type to another 33 genes were found to be up-regulated and in common to monocytes and the neuronal cells whereas 14 genes were in common to monocytes and olfactory cells; when genes from monocytes were up-regulated as compared to genes down-regulated in the other cell types, 39 genes were identified to be different in neuronal cells, whereas 52 genes were different in the olfactory cells. Our data suggest that C. pneumoniae-infected monocytes, olfactory neuroepithelia, and neuronal cells exhibit specific, but often disparate, changes in Alzheimer gene regulation. These gene changes appear to correlate with known pathologic changes observed in AD and further support the contention that infection with C. pneumoniae plays a role in AD pathogenesis.