Prognostic Value of Serum NPY Hypermethylation in Neoadjuvant Chemoradiotherapy for Rectal Cancer: Secondary Analysis of a Randomized Trial

Objectives: Long-term prevention of metastatic disease remains a challenge in locally advanced rectal cancer, and robust pretreatment prognostic factors for metastatic progression are lacking. We hypothesized that detecting circulating tumor-specific DNA (ctDNA) based on hypermethylation of the neuropeptide Y gene (meth-ctDNA) could be a prognostic marker in the neoadjuvant setting; we examined this in a secondary, explorative analysis of a prospective trial. Materials and Methods: Serum samples were prospectively collected in a phase III trial for locally advanced rectal cancer. Positivity for and fractional abundance of meth-ctDNA in baseline samples were estimated. Overall survival (OS) and the rate of distant metastases were compared between meth-ctDNA positive and negative patients; other prognostic factors were controlled for in multivariate Cox regression. Importance of quantitative load was examined by considering the fractional abundance of meth-ctDNA relative to total circulating DNA. Results: Baseline serum samples were available for 146 patients. In total, 30 patients had presence of meth-ctDNA, with no correlation with cT (P=0.8) or cN (P=0.6) stages. Median follow-up was 10.6 years for OS and 5.1 years for freedom from distant metastases. Patients with meth-ctDNA had significantly worse 5-year OS (47% vs. 69%), even when controlling for other prognostic factors (hazard ratio=2.08; 95% confidence interval, 1.23-1.51). This seemed mainly driven by disparity in the rate of distant metastases (55% vs. 72% at 5 y, P=0.01); hazard ratio=2.20 (95% confidence interval, 1.19-4.07, P=0.01) in multivariate analysis. Increased quantitative load was highly significant for worse outcomes. Conclusions: Meth-ctDNA could be a potential prognostic marker in the neoadjuvant setting and may, if validated, identify patients at increased risk of distant metastases

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