Christine J. Hammond

Chlamydia pneumoniae induces Alzheimer-like amyloid plaques in brains of BALB/c mice

Amyloid deposits resembling plaques found in Alzheimers 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 (Abeta) 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 Abeta1-42 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.

Chlamydia pneumoniae infection promotes the transmigration of monocytes through human brain endothelial cells

We have investigated the effects of Chlamydia pneumoniae on human brain endothelial cells (HBMECs) and human monocytes as a mechanism for breaching the blood‐brain barrier (BBB) in Alzheimers disease (AD). HBMECs and peripheral blood monocytes may be key components in controlling the entry of C. pneumoniae into the human brain. Our results indicate that C. pneumoniae infects blood vessels and monocytes in AD brain tissues compared with normal brain tissue. C. pneumoniae infection stimulates transendothelial entry of monocytes through HBMECs. This entry is facilitated by the up‐regulation of VCAM‐1 and ICAM‐1 on HBMECs and a corresponding increase of LFA‐1, VLA‐4, and MAC‐1 on monocytes. C. pneumoniae infection in HBMECs and THP‐1 monocytes up‐regulates monocyte transmigration threefold in an in vitro brain endothelial monolayer. In this way, C. pneumoniae infection in these cell types may contribute to increased monocyte migration and promote inflammation within the CNS resulting from infection at the level of the vasculature. Thus, infection at the level of the vasculature may be a key initiating factor in the pathogenesis of neurodegenerative diseases such as sporadic AD.

Immunohistological detection of Chlamydia pneumoniae in the Alzheimer's disease brain

BackgroundSporadic late-onset Alzheimers disease (AD) appears to evolve from an interplay between genetic and environmental factors. One environmental factor that continues to be of great interest is that of Chlamydia pneumoniae infection and its association with late-onset disease. Detection of this organism in clinical and autopsy samples has proved challenging using a variety of molecular and histological techniques. Our current investigation utilized immunohistochemistry with a battery of commercially available anti-C. pneumoniae antibodies to determine whether C. pneumoniae was present in areas typically associated with AD neuropathology from 5 AD and 5 non-AD control brains.ResultsImmunoreactivity 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 β 1-40 and 1-42 peptides.ConclusionsAnti-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 Alzheimers disease.

Inhibition of apoptosis in neuronal cells infected with Chlamydophila (Chlamydia) pneumoniae

BackgroundChlamydophila (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.ResultsSK-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. 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.ConclusionThese 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.

Detection of bacterial antigens and Alzheimer's disease-like pathology in the central nervous system of BALB/c mice following intranasal infection with a laboratory isolate of Chlamydia pneumoniae.

Pathology consistent with that observed in Alzheimer’s disease (AD) has previously been documented following intranasal infection of normal wild-type mice with Chlamydia pneumoniae (Cpn) isolated from an AD brain (96-41). In the current study, BALB/c mice were intranasally infected with a laboratory strain of Cpn, AR-39, and brain and olfactory bulbs were obtained at 1–4 months post-infection (pi). Immunohistochemistry for amyloid beta or Cpn antigens was performed on sections from brains of infected or mock-infected mice. Chlamydia-specific immunolabeling was identified in olfactory bulb tissues and in cerebrum of AR-39 infected mice. The Cpn specific labeling was most prominent at 1 month pi and the greatest burden of amyloid deposition was noted at 2 months pi, whereas both decreased at 3 and 4 months. Viable Cpn was recovered from olfactory bulbs of 3 of 3 experimentally infected mice at 1 and 3 months pi, and in 2 of 3 mice at 4 months pi. In contrast, in cortical tissues of infected mice at 1 and 4 months pi no viable organism was obtained. At 3 months pi, only 1 of 3 mice had a measurable burden of viable Cpn from the cortical tissues. Mock-infected mice (0 of 3) had no detectable Cpn in either olfactory bulbs or cortical tissues. These data indicate that the AR-39 isolate of Cpn establishes a limited infection predominantly in the olfactory bulbs of BALB/c mice. Although infection with the laboratory strain of Cpn promotes deposition of amyloid beta, this appears to resolve following reduction of the Cpn antigen burden over time. Our data suggest that infection with the AR-39 laboratory isolate of Cpn results in a different course of amyloid beta deposition and ultimate resolution than that observed following infection with the human AD-brain Cpn isolate, 96-41. These data further support that there may be differences, possibly in virulence factors, between Cpn isolates in the generation of sustainable AD pathology.

Infection of human monocytes, olfactory neuroepithelia, and neuronal cells with Chlamydia pneumoniae alters expression of genes associated with Alzheimer's disease

for BDNF expression. BDNF and TrkB were significantly reduced while TrkC andNT-3 were not altered. Moreover, BDNF was negatively related to AD-like tau phosphorylation. To proof the role of NGF and cholinergic innervation in this model, transgenic mice and controls were treated with intraventricular infusion of NGF or galantamine i.p. over two weeks. Treated animals displayed significantly increased numbers of BFCN and improved cognition. NGF treatment was superior over galantamine in this short-term treatment. Conclusions: Tau pathology plays an important role as it impairs axonal transport apparently involved in cholinergic degeneration. Here, we demonstrated the pivotal role of NTF in AD.

Evidence for an Infectious Etiology in Alzheimer's Disease

The possibility of an infectious etiology of several chronic diseases, including Alzheimer′s Disease, has long been debated. More than a century ago Alois Alzheimer studied neurological infection with Treponema pallidum, the causative agent of syphilis, a spirochete later associated with dementia (Noguchi and Moore, 1913). There are many chronic diseases for which there is strong evidence of an infectious etiology (see table 1), and numerous chronic diseases for which there is suspicion of infection as the etiologic agent for that disease (see table 2) (Tables 1 and 2 are adapted from a colloquium sponsored by the American Academy of Microbiology from June 2004). Koch’s postulates, that can, in some cases, provide absolute proof that a particular microorganism causes a particular disease, have been invaluable in the prevention and treatment of many diseases as well as in advancing microbiology. However, the postulates do not hold for most chronic diseases of microbial etiology, particularly those occurring late in life. Furthermore, they do not hold true for those of possible viral etiology, or for those that are multi-factorial in origin. In diseases of relatively old age, microbes acting earlier in life might operate by a “hit-andhide” mechanism, or could over time be present at an extremely low level, so that searches for the organism might not reveal the culprit until long after damage has been initiated. In viral diseases and for those involving unique organisms such as obligate intracellular bacteria (eg, Chlamydia), the postulates requiring isolation and growth in pure culture cannot be met completely as these organisms reproduce only within living cells. In multifactorial diseases, a causative organism might not be readily apparent, as other factors may be more prominent. However, absence of evidence is not proof of absence; in several cases when overwhelming experimental evidence was obtained, the pathogen concept had to be accepted even though it had met with great opposition initially. Two examples, among many, are the involvement of viruses in certain types of cancer such as human papillomavirus in cervical cancer, and of the bacterium Helicobacter pylori in stomach ulcers. Alzheimer′s Disease(AD) is a neurodegenerative disease that is considered to be the single most significant cause of dementia in the elderly (Keefover, 1996). There are two major categories of AD, familial and sporadic late-onset. The familial form of AD accounts for approximately 5% of total cases and usually presents in individuals in their 40’s and 50’s. This form of disease is caused by rare mutations in genes associated with β-amyloid

P3-223: Caspase activity is inhibited in neuronal cells infected with Chlamydia pneumoniae: Implications for apoptosis in Alzheimer’s disease

of A uptake 2) the requirement of A internalization for A -induced toxicity. Methods: Primary neurons cultured in three-compartmented dishes were treated with oligomeric fluorescein-conjugated A 1-42 exclusively in the distal axonor the cell body-containing compartments.A uptake and intracellular transport were analyzed by fluorescent microscopy and/or ELISA.The role of clathrinand caveolamediated endocytosis was analyzed in neurons infected with recombinant adenoviruses expressing HA-tagged wild-type dynamin-1, HA-tagged dynK44A mutant, or T7tagged clathrin hub. Neuronal apoptosis was determined by nuclear fragmentation and caspase activation. Results: A 1-42 was internalized mainly through the distal axons and retrogradely transported to cell bodies. Conversely, neurons treated with A 1-42 exclusively in the cell bodies showed very little A 1-42 internalization and anterograde transport . This result suggests that A 1-42 enters the cell by a regulated mechanism. Two main internalization pathways exist: namely clathrinand caveolae-mediated endocytosis. Both mechanisms require dynamin, but only the former is mediated by clathrin. Neurons expressing the dominat negative mutant of dynamin 1, showed a dramatic decrease in A 1-42 transport from distal axons to cell bodies. Conclusions: Our data suggest the oligomeric A 1-42 internalization is independent of clathrin. Our model is relevant to the death of basal forebrain cholinergic neurons in AD.

Chlamydia pneumoniae: An Etiologic Agent for Late-Onset Dementia

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 early-onset 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.

CELLULAR MODELING OF INFLAMMATION RELEVANT TO ALZHEIMER'S DISEASE: MOLECULAR AND CYTOKINE ANALYSIS OF HOST RESPONSES FOLLOWING CHLAMYDIAL INFECTION OF HUMAN THP-1 MONOCYTES

Background: Although the signature properties of Alzheimer’s disease (AD), such as the formation of amyloid plaques, activation of inflammatory responses, and hyperphosphorylation have been well studied, the explanation illustrating a clear relationship specifically between neuroinflammation and cognitive impairment has not yet been discovered. Thus in this article, we employed the McGill-R-Thy1-APP transgenic (Tg) rat model to observe for activation of inflammatory responses and the presence of cognitive decline. We hypothesize that early expression of neuroinflammatory signals will lead to future cognitive decline in the amyloid expressing animals. Methods:Microglial activation was measured using the ligand [F]PBR06 for PET, while cognitive performance was measured with the MWM task in 5 wild-type (Wt) and 8 Tg rats at baseline (BL; 11.5 months) and at follow-up (FU; 16 months). PBR-PET images were processed using the cerebellar grey matter as the reference region and cognitive measurement was calculated as the average times of day 3 and 4 of the water maze task. The baseline as well as the change (FU – BL) in the cognitive measurements were correlated at voxel level using “VoxelStats” toolbox to observe for the effect of group and PBR interaction on the change in the water maze performance. The PBR values were corrected for global cortical values, and t-statistical maps were generated to illustrate the regions of significance. Results:The association between baseline levels of cognition and inflammation was greater in the Tg than Wt rats in the right nucleus accumbens, whereas in the opposite was seen in the right inferior colliculus. The association between baseline levels of inflammation and change in cognition at follow-up, several regions including the left retrosplenial cortex, right hippocampus, and the right posterior commissure showed higher decrease in cognition of the Tg animals compared to the Wt (Figure 1). Conclusions:At baseline, there is no association between neuroinflammation and cognitive performance; however in more aged rats, baseline levels of PBR is able to predict cognitive decline. The results provide a framework that could potentially be applied in human studies focusing on the detrimental roles of neuroinflammation in AD. P2-054 CELLULAR MODELING OF INFLAMMATION RELEVANT TO ALZHEIMER’S DISEASE: MOLECULAR AND CYTOKINE ANALYSIS OF HOST RESPONSES FOLLOWING CHLAMYDIAL INFECTION OF HUMAN THP-1 MONOCYTES