Chlamydia pneumoniae Infection of Monocytes in vitro Stimulates Innate and Adaptive Immune Responses Relevant to those in Alzheimer\u27s Disease.

Background: Alzheimer\u27s disease (AD) is a progressive neurodegenerative disorder in which infection with Chlamydia pneumoniae (Cpn) has been associated. Cpn is an obligate intracellular respiratory pathogen that may enter the central nervous system (CNS) following infection and trafficking of monocytes through the blood-brain barrier. Following this entry, these cells may secrete pro-inflammatory cytokines and chemokines that have been identified in the AD brain, which have been thought to contribute to AD neurodegeneration. The objectives of this work were: (i) to determine if Cpn infection influences monocyte gene transcript expression at 48 hours post-infection and (ii) to analyze whether pro-inflammatory cytokines are produced and secreted from these cells over 24 to 120 hours post-infection. 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\u27s 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-1ß, 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\u27s disease

Mechanisms of Mouse Hepatitis Virus Entry into Cells

Background: Viruses can enter cells through several mechanisms, two common ones being clathrin-mediated and caveolin-mediated endocytosis. The clathrin pathway delivers viral particles to endosomes, with subsequent acidification of the endosome and endosome/lysosome fusion often a prerequisite for release of the viral genome into the cytoplasm. The caveolin-mediated pathway delivers virus initially into vesicles called caveosomes, which have a neutral pH, before viral uncoating occurs. Viral entry pathways can be examined by using various drugs to inhibit the different endocytosis pathways, as well as by siRNA technology to down-regulate expression of clathrin or caveolin proteins on the surface of host cells

Predicting Self-Management Behaviors in Familial Hypercholesterolemia Using an Integrated Theoretical Model: the Impact of Beliefs About Illnesses and Beliefs About Behaviors

Patients with familial hypercholesterolemia (FH) are at markedly increased risk of coronary artery disease. Regular participation in three self-management behaviors, physical activity, healthy eating, and adherence to medication, can significantly reduce this risk in FH patients. We aimed to predict intentions to engage in these self-management behaviors in FH patients using a multi-theory, integrated model that makes the distinction between beliefs about illness and beliefs about self-management behaviors. Methods: Using a cross-sectional, correlational design, patients (N = 110) diagnosed with FH from a clinic in Perth, Western Australia, self-completed a questionnaire that measured constructs from three health behavior theories: the common sense model of illness representations (serious consequences, timeline, personal control, treatment control, illness coherence, emotional representations); theory of planned behavior (attitudes, subjective norms, perceived behavioral control); and social cognitive theory (self-efficacy). Results: Structural equation models for each self-management behavior revealed consistent and statistically significant effects of attitudes on intentions across the three behaviors. Subjective norms predicted intentions for health eating only and self-efficacy predicted intentions for physical activity only. There were no effects for the perceived behavioral control and common sense model constructs in any model. Conclusions: Attitudes feature prominently in determining intentions to engage in self-management behaviors in FH patients. The prominence of these attitudinal beliefs about self-management behaviors, as opposed to illness beliefs, suggest that addressing these beliefs may be a priority in the management of FH

Infection of neuronal cells by Chlamydia pneumoniae and Herpes simplex virus type 1 alters expression of genes associated with Alzheimer’s disease

Several studies have suggested an infectious etiology for Alzheimer’s disease (AD). We have been investigating a potential role for both Chlamydia pneumoniae and Herpes simplex virus type 1 (HSV1) in the initiation of sporadic late-onset AD. Our current study focuses on investigation of gene expression using Alzheimer-specific Real-Time PCR microarrays on RNA derived from SKNMC human neuronal cells infected with C. pneumoniae and/or HSV1. There are distinct differences in the patterns of gene regulation by the two pathogens. For example, C. pneumoniae induces expression of genes involved in amyloid production and processing, such as β-amyloid precursor protein (APP), β-site APP-cleaving enzyme 1 (BACE1), a γ-secretase complex protein (nicastrin [NCSTN]), NEDD8 activating enzyme E1 (NAE1), as well as a mitochondria-associated protein (hydroxysteroid (17-β) dehydrogenase 10 [HSD17B10]), α-2-macroglobulin (A2M) and the metallopeptidase ADAM9. Conversely, HSV1 tends to down-regulate expression of many genes, including those encoding a component of the γ-secretase complex (anterior pharynx defective 1 homolog A [APH1A]), low density lipoprotein related proteins (LRP1, LRP6, and LRP8), β-synuclein (SNCB) and ubiquinols (UQCRC1, UQCRC2). Co-infection with C. pneumoniae and HSV-1 produced a greater down-regulation of gene expression than that seen with HSV1 alone for several genes, including APP-like proteins (APLP1, APLP2) and kinases (cell division cycle 2 protein [CDC2], cyclin-dependent kinase [CDK5] and CDC2-related kinase [CDKL1]). Our data indicate that both C. pneumoniae and HSV1 can modulate expression of genes associated with AD, and thus could contribute to AD pathology, however these two pathogens likely act via different pathways. Furthermore, for several genes, co-infection with both C. pneumoniae and HSV1 appears to exacerbate the changes in gene expression seen with HSV1 alone.https://digitalcommons.pcom.edu/posters/1007/thumbnail.jp

Autophagy and apoptotic genes implicated in Alzheimer’s disease are modulated following infection of neuronal cells with Chlamydia pneumoniae

Background: The focus of the current studies was to determine the relationship between the molecular mechanisms interconnecting autophagy and apoptosis following Chlamydia pneumoniae infection in neuronal cells. Dysfunctions in apoptosis and autophagy have been implicated in the neurodegeneration associated with Alzheimer’s disease (AD). Autophagy in AD pathogenesis has been shown to play a role in amyloid processing through the endosomal-lysosomal system. Apoptosis may contribute to the neuronal cell loss observed in AD; however, there is limited evidence of the apoptotic process proceeding to terminal completion. Although Aβ1-42 has been shown to induce apoptosis in neurons and may be an early factor in AD, our previous investigations demonstrated that neurons infected with Chlamydia pneumoniae are resistant to apoptosis, and that Aβ1-42 is induced following this infection. Thus, these studies address infection as an initiator/trigger or inhibitor for the processes of autophagy and apoptosis observed in Alzheimer’s disease. Methods: SKNMC neuronal cells obtained from ATCC were infected with the AR39 strain of Chlamydia pneumoniae at an MOI=1 for 24, 48, and 72hrs and were analyzed using Real-time PCR arrays from SABiosciences specific for autophagy and apoptosis genetic markers. Results: Some major genes associated with apoptosis such as BID, DAPK1, TP53, TP73 were down regulated by 72hrs post-infection. Genes associated with the regulation of autophagic vacuole formation such as ATG3, ATG4B, ATG4C, ATG9A, ATG9B, ATG12, IRGM, and BECN1 were up-regulated within 72hrs post-infection. With regards to genes involved with co-regulation of autophagy and apoptosis, BNIP3 was significantly up-regulated within 48-72hrs post-infection. Of the genes linking autophagosomes to lysosomes, FAM176A was up-regulated throughout 24-72hrs post-infection. Conclusions: Modulation of autophagy and apoptosis genes occurs in neuronal cells at 24, 48, and 72hrs post- infection with Chlamydia pneumoniae. These genetic changes lead to dysfunction in these basic cellular processes; dysfunction in these processes has been shown to contribute to the neuropathology of late-onset Alzheimer’s disease. This work will allow future studies to further focus on the apoptotic and autophagic pathways to better understand how a pathogen such as Chlamydia pneumoniae plays a role in the development of late-onset Alzheimer’s disease.https://digitalcommons.pcom.edu/posters/1009/thumbnail.jp

Changes in Expression of Genes Associated with Autophagy and Apoptosis in Neuronal Cells Infected with HSV-1may Suggest Infection-induced Mechanisms of Neurodegeneration

Background:This study investigates the potential role of herpes simplex virus type 1 (HSV-1) in the pathogenesis of neurodegenerative disorders, such as Alzheimer’s disease (AD), by exploring changes in gene expression related to antiviral immunity and the autophagic pathway. Autophagy is a process that recycles organelles and proteins to create more energy for the cell. This pathway has been linked to neurodegeneration, as malfunctions in the completion of this process lead to a decline in overall cellular health and function. Interestingly, HSV-1 has been shown to block the completion of autophagy, which would potentially contribute to the cytopathic changes observed in AD

Infection with Chlamydia Pneumoniae Alters Calcium-associated Gene Regulation and Processes in Neuronal Cells and Monocytes: Implications for Alzheimer’s Disease

Background: First proposed by Khachaturian in 1994, the calcium hypothesis postulates that sustained disturbance of intracellular calcium is the leading cause of neurodegenerative disorders. Studies showing alteration in calcium signaling in both sporadic and familial Alzheimer’s disease (AD) support this hypothesis. Intracellular calcium signaling is tightly regulated in time, intensity, and space, and is responsible for a variety of neuronal functions. Calcium influx from the extracellular environment modulates calcium levels, as do intracellular stores in the endoplasmic reticulum. The focus of this study was to test various calcium related genes in both monocytes and neuronal cells. Previous studies have shown that cells infected with Chlamydia pneumoniae (Cpn) exhibit altered protein processing, such as amyloid and tau modification, consistent with those found in AD. We expect to see significant alterations in calcium genes, as well as their protein products in Cpn infected cells. Every calcium gene has a unique function in the cell. Determining which genes are up or down regulated following infection may provide insight into how the neurodegeneration process observed in AD is initiated by Cpn infections

Herpes Simplex Virus 1 and Chlamydophila (Chlamydia) pneumoniae promote Ab 1-42 amyloid processing in murine astrocytes linking an infectious process to Alzheimer\u27s disease

Background: Several studies have suggested an infectious etiology for Alzheimer\u27s disease (AD). Previously, our laboratory identified Chlamydia pneumoniae (Cpn) from autopsied sporadic AD brains, as well as developed a BALB/c mouse model that demonstrated infection-induced amyloid plaques similar to those found in AD. Hypothesis: We propose that an additional pathogen such as herpes simplex virus type 1 (HSV1), also may be a contributing factor in toin the pathology seen in AD. HSV1, in addition to Cpn, may be triggering the abnormal cleavage of the beta amyloid precursor protein (bAPP) into Ab1-42 , thereby contributing to amyloid plaque formation. Our current study examines amyloid processing following infection of primary and C8-DIA murine astrocytes with Cpn and HSV1. Materials and Methods: Immunocytochemistry and western analysis was used to analyze the outcome of infection by these two pathogens. Results: Cpn infection resulted in an increase in cytoplasmic labeling of Ab 1-42 relative to uninfected cells, while increased nuclear labeling of Ab 1-42 was observed following HSV1 infection. Co-infections with Cpn and HSV1 resulted in amyloid labeling resembling that of HSV1 infection alone, though Ab 1-42 labeling appeared decreased specifically in Cpn-infected cells of the co-infected monolayers. Conclusions: These data suggest that infection of astrocytic cells by HSV1 and (Cpn) alter the processing of bAPP, thereby producing Ab1-42. Therefore, these studies, inaddition to the previous research reported by our laboratory, support an emerging linkage of the infectious processs to the neuropathology characteristic of Alzheimer\u27s disease.https://digitalcommons.pcom.edu/posters/1008/thumbnail.jp

Analysis of Chlamydia pneumoniae-infected monocytes following incubation with a novel peptide, acALY18, implicates the inflammasome in clearance of infection

Chlamydia pneumoniae infection may be a trigger for the pathology observed in sporadic lateonset Alzheimer’s disease as a function of initiating neuroinflammation following entry of the organism into the brain. We have hypothesized that one entry mechanism may be by bloodborne infected monocytes trafficking the infection into the brain. This study focuses on infection of monocytes in vitro followed by analysis using immunofluorescence labeling and RT-PCR-microarray techniques. The microarrays utilized consisted of an Alzheimer’s disease pathway array and an innate and adaptive immunity array from SAbiosciences. Analysis by real time PCR for both gene arrays was performed on uninfected and C. pneumoniae-infected THP1 monocytes at 48 hr post-infection. In addition, we analyzed innate and adaptive immunity gene regulation changes following treatment of infected cells with a unique peptide, acALY18, derived from the endogenously expressed endoplasmic reticulum protein TRPC1. The peptide appears to stimulate the innate immune system through activation of the inflammasome. C. pneumoniae prominently infected THP1 monocytes at 24-48hr. Numerous large inclusions were identified using specific chlamydial monoclonal antibodies. Monocyte gene expression changes induced by infection with C. pneumoniae revealed significant up-regulation of 45 genes in the Alzheimer’s disease pathway. These included genes involved in: b-amyloid processing and clearance, apoptosis, proteases and protein kinases, and lipid metabolism. In contrast, infection resulted in significant changes in 30 genes governing innate and adaptive immunity including those for: the inflammatory response, host defense against bacteria, cytokines, chemokines, and an antibacterial humoral response. Intriguingly, following incubation of C. pneumoniae-infected cells with the acALY18 peptide (25-50nM) at 24hr post-infection, there was significant clearance of the organism from the monocytes as well as up-regulation of 38 genes. Our data suggest that C. pneumoniae infection of monocytes has a profound effect on gene regulation for both innate and adaptive immunity and for Alzheimer’s disease. Stimulating the innate immune response using the novel peptide, acALY18, promotes clearance of C. pneumoniae from infected monocytes; thereby implicating the inflammasome as a key component in eradicating this infection.https://digitalcommons.pcom.edu/posters/1005/thumbnail.jp

Analysis of autophagy and inflammasome regulation in neuronal cells and monocytes infected with Chlamydia pneumoniae: Implications for Alzheimer’s disease

Objectives: Our laboratory has been studying the role of infection with the obligate intracellular bacterium 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. As such, we are analyzing how this infection can promote changes in autophagy and inflammasome gene regulation as both have been shown to be altered in LOAD. Methods: Human SKNMC neuronal cells and THP1 monocytes were infected in vitro for 24-72 hrs with a laboratory strain of Chlamydia pneumoniae followed by RNA extraction, cDNA synthesis and analysis using Real-Time PCR microarrays for autophagy and inflammasome genes. Results: Gene expression for autophagy and inflammasome pathways was altered dramatically following infection. Genes encoding for co-regulation of autophagy, apoptosis, and the cell cycle that were significantly changed included: BCL2L1, FAS, PIK3CG, APP, and TP53. In addition, ATG3, and GABARAP, genes encoding for protein transport & ubiquitination and autophagic vacuole formation were significantly deregulated. Of the inflammasome genes, 4 NOD-like receptor genes were significantly up-regulated. IL-1beta, AIM2, CCL2, and CCL7 genes were all dramatically up-regulated in monocytes during the 72 hrs of infection. Conclusions: Our data suggest that Chlamydia pneumoniae-infected human SKNMC neuronal cells and THP1 monocytes exhibit specific changes in gene regulation for both autophagy and inflammasome pathways. These gene changes appear to correlate with pathologic changes previously reported in AD and further support the contention that infection with Chlamydia pneumoniae plays a role in LOAD pathogenesis.https://digitalcommons.pcom.edu/posters/1001/thumbnail.jp