Astrocytes Infected with Chlamydia pneumonia Alter Amyloid Processing Implicated in Alzheimer’s Disease

Alzheimer’s Disease (AD) is a chronic, progressive neurodegenerative disease whose pathogenesis centers around the abnormal processing of amyloid precursor protein (APP) by proteases, resulting in the formation of neuritic plaques composed of toxic, insoluble fragments of amyloid protein (Aβ), including Aβ1-40 and Aβ1-42. Previously, our laboratory identified Chlamydia pneumoniae (Cpn) in autopsied sporadic AD brains. Additionally, an infection based animal model was developed using BALB/c mice that were intranasally inoculated with Cpn, in which the deposition of amyloid was consistent with that observed in the human AD brain. These studies have led to the pathogen hypothesis of AD that implicates Cpn as a trigger for the cleavage of APP into Aβ1-40 and Aβ1-42. Objective: Several studies have demonstrated the presence of astrocytes surrounding neuritic plaques within the AD brain; therefore, we speculate that astrocytes may be specifically involved in the pathological processes leading to Aβ deposition. This investigation addresses if an in vitro Cpn infection of human astrocytes affects processing of the ß amyloid precursor protein (ßAPP) and the enzyme ß APP cleaving enzyme-1 (BACE1), a type 1 transmembrane aspartyl protease directly involved in the processing of APP to Aβ and implicated in numerous neurodegenerative diseases, such as traumatic brain injury. Methods: Human astrocytes (CCF-STTG1) were infected in vitro with the respiratory strain AR39 Cpn (MOI=1). Analysis of protein levels for Aβ and the enzyme BACE1 post-infection was detected by immunocytochemistry and captured with the Olympus Confocal FV1000 microscope. Results: Amyloid processing in infected astrocytes was altered relative to that of uninfected astrocytes. BACE1 immunolabeling appeared more diffuse in the infected astrocytes as compared to membrane-localized BACE1 in the uninfected astrocytes. Conclusions: Neurons have been presumed to be the primary source of beta-amyloid peptides in AD brains; however, when astrocytes are activated, as occurs during infection with Cpn, astrocytic beta-amyloid generation may contribute to amyloid plaque formation. These data imply that infection of human astrocytes with Cpn affects the processing of ßAPP through altering the localization of BACE1 protein from the membrane to the cytoplasm. These data suggest an activation of BACE1 in the processing of amyloid by astrocytes as a major contributor to the neurotoxic amyloid deposition linked to pathology observed in AD

Astrocytes Infected with Chlamydia Pneumoniae Demonstrate Altered Expression and Activity of Secretases Involved in the Generation of Β-amyloid Found in Alzheimer Disease

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. 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

Assessment of Attitudes and Knowledge of Osteopathic Manipulative Medicine Amongst Neurosurgeons

Introduction: Osteopathic Manipulative Medicine (OMM) is highly researched and integrated into many medical and surgical subspecialties. Though OMM has been shown to have benefits in treating non-specific cervical and lower back pain and managing post-operative pain, it has rarely been integrated or studied in the neurosurgical community. Method: Using SurveyMonkey, a survey was dispersed to physicians of the American Association of Neurological Surgeons (AANS). Information collected included the provider’s type of practice, degree type (MD vs. DO), familiarity with the tenets of OMM, frequency of referral/use of OMM, comfort level with OMM as a non-surgical pre-op option, a post-op pain management option, and a post-op rehabilitation option. It also assessed surgeons\u27 interest in more research on OMM in the neurosurgical field and the likelihood they would integrate OMM into their practice if given peer-reviewed research about OMM in their patient population. Responses from MDs and DOs were averaged and, when appropriate, were compared using a two-tailed T-test, with statistical significance defined as p \u3c 0.05. Results: Of the 6503 neurosurgeons surveyed, 133 submitted responses, and 121 met inclusion criteria. This cohort consisted of 114 Allopathic physicians (MDs) and 7 Osteopathic physicians (DOs) (94.2% and 5.8%, respectively). On average, neurosurgeons reported referring patients for OMM less than once per year (average = 1.36 ± 0.79, where ‘1’ = never and ‘2’ = once per year), with 79.33% responding ‘never.’ When comparing DO to MD neurosurgeons, DOs ranked both their familiarity with the tenets of osteopathic medicine (4.57 ± 0.53 vs. 2.47 ± 1.02, respectively) and applications of OMM in their practice (3.29 ± 1.38 vs. 1.97 ± 1.05, respectively) significantly higher. DO neurosurgeons were significantly more comfortable than MDs in recommending both pre-operative and post-operative care as well as non-operative pain management (pre-operative, average = 3.71 vs. 2.38, p=0.0037; post-operative, average = 3.29 ± 1.25 vs. 2.23 ± 1.04, p = 0.011; pain management, average = 4.00 ± 0.82, 2.60 ± 1.10 p = 0.0013). DOs and MDs show no statistical difference when assessed for interest in future research on the application of OMM within neurosurgery and were self-rated as “somewhat Interested”(average 3.14, 3 - somewhat interested). If presented with research demonstrating the positive applications of OMM relevant research to the management of neurosurgery patients, DOs and MDs trended towards being “likely” to integrate OMM into their practice (average = 3.43 ± 1.52, 3.83 ± 0.89 p = 0.2666 ). Discussion: ln general, osteopathic physicians reported greater comfort with OMM, likely related to DO’s greater familiarity with the topic due to curriculum differences in medical school training compared to their MD colleagues. Though MD and DO neurosurgeons infrequently utilize or refer to OMM in their practice, both groups are interested in seeing more research on OMM in the neurosurgical field and are open to utilizing evidence-based OMM in patient management

Chlamydia pneumoniae: An Etiologic Agent for Late-Onset Dementia

The disease known as late-onset Alzheimer\u27s disease is a neurodegenerative condition recognized as the single most common form 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\u27s 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 pathoge

Minimally Invasive In Vivo Real-Time Identification of Human Astrocytoma with Sulforhodamine 101

Abstract and poster under embargo until May 31, 2019

Expanding the Diversity of Mycobacteriophages: Insights into Genome Architecture and Evolution

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists

Delta-Aminolevulinic Acid-Mediated Photodiagnoses in Surgical Oncology: A Historical Review of Clinical Trials.

Fluorescence imaging is an emerging clinical technique for real-time intraoperative visualization of tumors and their boundaries. Though multiple fluorescent contrast agents are available in the basic sciences, few fluorescence agents are available for clinical use. Of the clinical fluorophores, delta aminolevulinic acid (5ALA) is unique for generating visible wavelength tumor-specific fluorescence. In 2017, 5ALA was FDA-approved for glioma surgery in the United States. Additionally, clinical studies suggest this agent may have utility in surgical subspecialties outside of neurosurgery. Data from dermatology, OB/GYN, urology, cardiothoracic surgery, and gastrointestinal surgery show 5ALA is helpful for intraoperative visualization of malignant tissues in multiple organ systems. This review summarizes data from English-language 5ALA clinical trials across surgical subspecialties. Imaging systems, routes of administration, dosing, efficacy, and related side effects are reviewed. We found that modified surgical microscopes and endoscopes are the preferred imaging devices. Systemic dosing across surgical specialties range between 5 and 30 mg/kg bodyweight. Multiple studies discussed potential for skin irritation with sun exposure, however this side effect is infrequently reported. Overall, 5ALA has shown high sensitivity for labeling malignant tissues and providing a means to visualize malignant tissue not apparent with standard operative light sources

Intratympanic corticosteroids in Ménière's disease: A mini-review

This article reviews the effectiveness of intratympanic corticosteroids for vertigo control in Ménière's disease at 2-years follow-up according to the guidelines expressed by the American Academy of Otolaryngology-Head & Neck Surgery. Despite the increased use of intratympanic corticosteroids for vertigo control in Ménière's disease there is debate as to their effectiveness, particularly compared to gentamicin. Even so, after just a single course of injections, corticosteroids can reliably provide complete vertigo control (Class A) at 2-years in about 50% of cases as indicated in a recent double-blind randomized controlled clinical trial (Patel et al., 2016). But the effectiveness of intratympanic corticosteroids truly increases when treatment is provided ‘as-needed’, whereby complete vertigo control is established in up to 91% of cases. On the basis of available literature, there is good evidence to recommend the use of intratympanic steroid treatment for vertigo control in Ménière's disease, but patients must be monitored for non-response. The rationale for treating patients as-needed and the possible reasons for corticosteroid non-response are discussed

A Novel HLA-A*0201 Restricted Peptide Derived from Cathepsin G Is an Effective Immunotherapeutic Target in Acute Myeloid Leukemia

Immunotherapy targeting aberrantly expressed leukemia associated antigens (LAA) has shown promise in the management of acute myeloid leukemia (AML). However, because of the heterogeneity and clonal evolution that is a feature of myeloid leukemia, targeting single peptide epitopes has had limited success, highlighting the need for novel antigen discovery. In this study, we characterize the role of the myeloid azurophil granule protease cathepsin G (CG) as a novel target for AML immunotherapy

Expanding the diversity of mycobacteriophages: Insights into genome architecture and evolution

Mycobacteriophages are viruses that infect mycobacterial hosts such as Mycobacterium smegmatis and Mycobacterium tuberculosis. All mycobacteriophages characterized to date are dsDNA tailed phages, and have either siphoviral or myoviral morphotypes. However, their genetic diversity is considerable, and although sixty-two genomes have been sequenced and comparatively analyzed, these likely represent only a small portion of the diversity of the mycobacteriophage population at large. Here we report the isolation, sequencing and comparative genomic analysis of 18 new mycobacteriophages isolated from geographically distinct locations within the United States. Although no clear correlation between location and genome type can be discerned, these genomes expand our knowledge of mycobacteriophage diversity and enhance our understanding of the roles of mobile elements in viral evolution. Expansion of the number of mycobacteriophages grouped within Cluster A provides insights into the basis of immune specificity in these temperate phages, and we also describe a novel example of apparent immunity theft. The isolation and genomic analysis of bacteriophages by freshman college students provides an example of an authentic research experience for novice scientists. © 2011 Hatfull et al