Activation of Endogenous Retrovirus, Brain Infections and Environmental Insults in Neurodegeneration and Alzheimer's Disease.

Chronic neurodegenerative diseases are complex, and their pathogenesis is uncertain. Alzheimer's disease (AD) is a neurodegenerative brain alteration that is responsible for most dementia cases in the elderly. AD etiology is still uncertain; however, chronic neuroinflammation is a constant component of brain pathology. Infections have been associated with several neurological diseases and viruses of the Herpes family appear to be a probable cause of AD neurodegenerative alterations. Several different factors may contribute to the AD clinical progression. Exogeneous viruses or other microbes and environmental pollutants may directly induce neurodegeneration by activating brain inflammation. In this paper, we suggest that exogeneous brain insults may also activate retrotransposons and silent human endogenous retroviruses (HERVs). The initial inflammation of small brain areas induced by virus infections or other brain insults may activate HERV dis-regulation that contributes to neurodegenerative mechanisms. Chronic HERV activation in turn may cause progressive neurodegeneration that thereafter merges in cognitive impairment and dementia in genetically susceptible people. Specific treatment for exogenous end endogenous pathogens and decreasing pollutant exposure may show beneficial effect in early intervention protocol to prevent the progression of cognitive deterioration in the elderly.

MicroRNA expression profiling with a droplet digital PCR assay enables molecular diagnosis and prognosis of cancers of unknown primary.

Metastasis is responsible for the majority of cancer-related deaths. Particularly, challenging is the management of metastatic cancer of unknown primary site (CUP), whose tissue of origin (TOO) remains undetermined even after extensive investigations and whose therapy is rather unspecific and poorly effective. Molecular approaches to identify the most probable TOO of CUPs can overcome some of these issues. In this study, we applied a predetermined set of 89 microRNAs (miRNAs) to infer the TOO of 53 metastatic cancers of unknown or uncertain origin. The miRNA expression was assessed with droplet digital PCR in 159 samples, including primary tumors from 17 tumor classes (reference set) and metastases of known and unknown origin (test set). We combined two different statistical models for class prediction to obtain the most probable TOOs: the nearest shrunken centroids approach of Prediction Analysis of Microarrays (PAMR) and the least absolute shrinkage and selection operator (LASSO) models. The molecular test was successful for all formalin-fixed paraffin-embedded samples and provided a TOO identification within 1 week from the biopsy procedure. The most frequently predicted origins were gastrointestinal, pancreas, breast, lung, and bile duct. The assay was applied also to multiple metastases from the same CUP, collected from different metastatic sites: The predictions showed a strong agreement, intrinsically validating our assay. The final CUPs' TOO prediction was compared with the clinicopathological hypothesis of primary site. Moreover, a panel of 13 miRNAs proved to have prognostic value and be associated with overall survival in CUP patients. Our study demonstrated that miRNA expression profiling in CUP samples could be employed as diagnostic and prognostic test. Our molecular analysis can be performed on request, concomitantly with standard diagnostic workup and in association with genetic profiling, to offer valuable indications about the possible primary site, thereby supporting treatment decisions.

Genetic Characterization of Cancer of Unknown Primary Using Liquid Biopsy Approaches.

Cancers of unknown primary (CUPs) comprise a heterogeneous group of rare metastatic tumors whose primary site cannot be identified after extensive clinical-pathological investigations. CUP patients are generally treated with empirical chemotherapy and have dismal prognosis. As recently reported, CUP genome presents potentially druggable alterations for which targeted therapies could be proposed. The paucity of tumor tissue, as well as the difficult DNA testing and the lack of dedicated panels for target gene sequencing are further relevant limitations. Here, we propose that circulating tumor cells (CTCs) and circulating tumor DNA (ctDNA) could be used to identify actionable mutations in CUP patients. Blood was longitudinally collected from two CUP patients. CTCs were isolated with CELLSEARCH® and DEPArrayTM NxT and Parsortix systems, immunophenotypically characterized and used for single-cell genomic characterization with Ampli1TM kits. Circulating cell-free DNA (ccfDNA), purified from plasma at different time points, was tested for tumor mutations with a CUP-dedicated, 92-gene custom panel using SureSelect Target Enrichment technology. In parallel, FFPE tumor tissue was analyzed with three different assays: FoundationOne CDx assay, DEPArray LibPrep and OncoSeek Panel, and the SureSelect custom panel. These approaches identified the same mutations, when the gene was covered by the panel, with the exception of an insertion in APC gene. which was detected by OncoSeek and SureSelect panels but not FoundationOne. FGFR2 and CCNE1 gene amplifications were detected in single CTCs, tumor tissue, and ccfDNAs in one patient. A somatic variant in ARID1A gene (p.R1276∗) was detected in the tumor tissue and ccfDNAs. The alterations were validated by Droplet Digital PCR in all ccfDNA samples collected during tumor evolution. CTCs from a second patient presented a pattern of recurrent amplifications in ASPM and SEPT9 genes and loss of FANCC. The 92-gene custom panel identified 16 non-synonymous somatic alterations in ccfDNA, including a deletion (I1485Rfs∗19) and a somatic mutation (p. A1487V) in ARID1A gene and a point mutation in FGFR2 gene (p.G384R). Our results support the feasibility of non-invasive liquid biopsy testing in CUP cases, either using ctDNA or CTCs, to identify CUP genetic alterations with broad NGS panels covering the most frequently mutated genes.

Unraveling the role of microRNA/isomiR network in multiple primary melanoma pathogenesis.

Malignant cutaneous melanoma (CM) is a potentially lethal form of skin cancer whose worldwide incidence has been constantly increasing over the past decades. During their lifetime, about 8% of CM patients will develop multiple primary melanomas (MPMs), usually at a young age and within 3 years from the first tumor/diagnosis. With the aim of improving our knowledge on MPM biology and pathogenesis, we explored the miRNome of 24 single and multiple primary melanomas, including multiple tumors from the same patient, using a small RNA-sequencing approach. From a supervised analysis, 22 miRNAs were differentially expressed in MPM compared to single CM, including key miRNAs involved in epithelial-mesenchymal transition. The first and second melanoma from the same patient presented a different miRNA profile. Ten miRNAs, including miR-25-3p, 149-5p, 92b-3p, 211-5p, 125a-5p, 125b-5p, 205-5p, 200b-3p, 21-5p, and 146a-5p, were further validated in 47 single and multiple melanoma samples. Pathway enrichment analysis of miRNA target genes revealed a more differentiated and less invasive status of MPMs compared to CMs. Bioinformatic analyses at the miRNA isoform (isomiR) level detected a panel of highly expressed isomiRs belonging to miRNA families implicated in human tumorigenesis, including miR-200, miR-30, and miR-10 family. Moreover, we identified hsa-miR-125a-5p|0|-2 isoform as tenfold over-represented in melanoma than the canonical form and differentially expressed in MPMs arising in the same patient. Target prediction analysis revealed that the miRNA shortening could change the pattern of target gene regulation, specifically in genes implicated in cell adhesion and neuronal differentiation. Overall, we provided a putative and comprehensive characterization of the miRNA/isomiR regulatory network of MPMs, highlighting mechanisms of tumor development and molecular features differentiating this subtype from single melanomas.

Method for the Detection of the Cleaved Form of Shiga Toxin 2a Added to Normal Human Serum.

The pathogenesis of Escherichia coli-induced hemolytic uremic syndrome (eHUS) caused by infections with pathogenic Shiga toxin (Stx) producing E. coli (STEC) is centered on bacterial (e.g., Stx) and host factors (circulating cells, complement system, serum proteins) whose interaction is crucial for the immediate outcome and for the development of this life-threatening sequela. Stx2a, associated to circulating cells (early toxemia) or extracellular vesicles (late toxemia) in blood, is considered the main pathogenic factor in the development of eHUS. Recently, it was found that the functional properties of Stx2a (binding to circulating cells and complement components) change according to modifications of the structure of the toxin, i.e., after a single cleavage of the A subunit resulting in two fragments, A1 and A2, linked by a disulfide bridge. Herein, we describe a method to be used for the detection of the cleaved form of Stx2a in the serum of STEC-infected or eHUS patients. The method is based on the detection of the boosted inhibitory activity of the cleaved toxin, upon treatment with reducing agents, on a rabbit cell-free translation system reconstituted with human ribosomes. The method overcomes the technical problem caused by the presence of inhibitors of translation in human serum that have been stalled by the addition of RNAase blockers and by treatment with immobilized protein G. This method, allowing the detection of Stx2a at concentrations similar to those found by ELISA in the blood of STEC-infected patients, could be a useful tool to study the contribution of the cleaved form of Stx2a in the pathogenesis of eHUS.

Defining the Prognostic Role of MicroRNAs in Cutaneous Melanoma.

Breslow thickness (BT) is the most important histopathologic factor for primary melanoma staging. BT determines the margins for wide local excision whether sentinel lymph node biopsy should be performed and subsequent melanoma staging, and patient management. The correct determination of a 0.8-mm cutoff in melanoma is important for pathologists because discrepancies leading to a change in stage can have significant clinical implications, including incorrect and/or inappropriate prognostic information, investigation, management, and follow-up. Difficulties in BT determination are mostly represented by the presence of regression or melanoma associated with a pre-existing nevus. This study aimed at investigating a molecular parameter, namely microRNA (miRNA) expression, in reference to BT assessment. Melanoma cell proliferation is influenced by miRNA dysregulation. Indeed, some miRNAs sustain and induce proliferative signals or repress growth-suppressive pathways, thereby promoting melanoma carcinogenesis. To identify the miRNAs correlating with BT, we analyzed our global miRNA expression data of 20 thin melanomas and identified two potential candidates, miR-21-5p and miR-146a-5p. We assessed the expression of these two specific miRNAs in 90 archive formalin-fixed and paraffin-embedded samples of superficially spreading melanomas (SSMs) and 25 nodular melanomas from two independent cohorts and correlated the individual and combined miRNA expression with BT and other tumor characteristics. The individually normalized expression of miR-21-5p and miR-146a-5p showed a highly significant and linear correlation with BT in SSM, and their combined expression value was more strongly correlated (Pearson's r = 0.799, 95% CI = 0.71-0.86) than their individual expressions. This correlation was not significant in nodular melanoma. In SSM, we observed that the combined miRNA expression above or below 1.5 was significantly associated with overall survival and successfully identified all patients with relapsing SSM. We concluded that the combined assessment of miR-21-5p and miR-146a-5p expression in superficially spreading melanoma, in association with BT measurement, could aid pathologists in SSM staging.

Impaired Innate Immunity Mechanisms in the Brain of Alzheimer's Disease.

Among environmental factors likely associated with Alzheimer's disease (AD), persistent virus infections, and age-related progressive decline of immune competence might play a pivotal role. However, AD antimicrobial brain immune responses are poorly investigated. The present study focused on genes involved in antimicrobial defenses, especially against virus infections, in the AD brain. In particular, mRNA levels of IRF7, MED23, IL28B, and IFN-α genes were analyzed in hippocampus and temporal cortex brain samples from AD and non-demented controls. All subjects were also genotyped for APOE ε, IRF7, MED23, and IL28B gene polymorphisms. Most AD patients showed decreased mRNA levels of all investigated genes in the hippocampus and temporal cortex. However, a small group of AD patients showed increased hippocampal mRNA expression of MED23, IL28B, and IFN-α. mRNA levels of MED23, IL28B, IFN-α from the hippocampus and those of MED23 from the temporal cortex were further decreased in APOE ε4 allele AD carriers. Moreover, rs6598008 polymorphism of IRF7 was significantly associated with decreased hippocampal expression of IRF7, MED23, IL28B, and IFN-α. These findings suggest that AD brains show impaired innate antimicrobial gene expression profiles, and individual genetic makeup, such as positivity for the APOE ε4 and IRF7 A alleles, might affect brain immune efficiency.

Particulate Shiga Toxin 2 in Blood is Associated to the Development of Hemolytic Uremic Syndrome in Children.

Hemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children (< 3 years), is mainly related to Shiga toxins (Stx)-producing Escherichia coli (STEC) infections. STEC are confined to the gut resulting in hemorrhagic colitis, whereas Stx are delivered in blood to target kidney and brain, with unclear mechanisms, triggering HUS in 5 to 15% of infected children. Stx were found on circulating cells, free in sera (soluble Stx) or in blood cell-derived microvesicles (particulate Stx), whereby the relationship between these forms of circulating toxins is unclear. Here, we have examined 2,846 children with bloody diarrhea and found evidence of STEC infection in 5%. Twenty patients were enrolled to study the natural course of STEC infections before the onset of HUS. In patients, Stx were found to be associated to circulating cells and/or free and functionally active in sera. In most children, Stx were bound to neutrophils when high amounts of toxins were found in feces. Time-course analysis showed that Stx increased transiently in patients' sera while the decrease of toxin amount on leukocytes was observed. Notably, patients who recovered (85%) displayed different settings than those who developed HUS (15%). The distinctive feature of the latter group was the presence in blood of particulate Stx2 (Stx2 sedimented at g-forces corresponding to 1 µm microvesicles) the day before diagnosis of HUS, during the release phase of toxins from circulating cells. This observation strongly suggests the involvement of blood cell-derived particulate Stx2 in the transition from hemorrhagic colitis to HUS.

KRAS and ERBB-family genetic alterations affect response to PD-1 inhibitors in metastatic nonsquamous NSCLC.

BACKGROUND: Programmed cell death 1 (PD-1) and PD-ligand 1 (PD-L1) inhibitors represent novel therapeutic options for advanced non-small cell lung cancer (NSCLC). However, approximately 50% of patients do not benefit from therapy and experience rapid disease progression. PD-L1 expression is the only approved biomarker of benefit to anti-PD-1/PD-L1 therapy. However, its weakness has been evidenced in many studies. More recently, tumor mutational burden (TMB) has proved to be a suitable biomarker, but its calculation is difficult to obtain for all patients. METHODS: We tested specific NSCLC genetic alterations as potential immunotherapy biomarkers. Tumor DNA was obtained from advanced NSCLC patients treated with anti-PD-1 monoclonal antibody nivolumab (n = 44) or pembrolizumab (n = 3). The mutational status of 22 genes was assessed by targeted next-generation sequencing and the association with survival was tested in uni- and multivariate models. The association between gene mutations and clinical benefit was also investigated. RESULTS: The most frequently mutated genes were TP53 (49%), KRAS (43%), ERBB2 (13%), SMAD4 (13%), DDR2 (13%), STK11 (9%), ERBB4 (6%), EGFR (6%), BRAF (6%), and MET (6%). We confirmed that KRAS mut patients have a better response to PD-1 inhibitors, showing a longer progression-free survival (PFS) and overall survival (OS) than KRAS wt patients. In addition, we observed that patients with ERBB-family mutations, including EGFR, ERBB2, and ERBB4 all failed to respond to PD-1 antibodies, independently of KRAS status. CONCLUSIONS: This study suggests that the analysis of KRAS and ERBB-family gene mutational status is valuable when assessing the clinical practice for the selection of NSCLC patients to treat with PD-1 inhibitors.

Interplay between small and long non-coding RNAs in cutaneous melanoma: a complex jigsaw puzzle with missing pieces.

The incidence of cutaneous melanoma (CM) has increased in the past few decades. The biology of melanoma is characterized by a complex interaction between genetic, environmental and phenotypic factors. A greater understanding of the molecular mechanisms that promote melanoma cell growth and dissemination is crucial to improve diagnosis, prognostication, and treatment of CM. Both small and long non-coding RNAs (lncRNAs) have been identified to play a role in melanoma biology; microRNA and lncRNA expression is altered in transformed melanocytes and this in turn has functional effects on cell proliferation, apoptosis, invasion, metastasis, and immune response. Moreover, specific dysregulated ncRNAs were shown to have a diagnostic or prognostic role in melanoma and to drive the establishment of drug resistance. Here, we review the current literature on small and lncRNAs with a role in melanoma, with the aim of putting into some order this complex jigsaw puzzle.

The structure of the Shiga toxin 2a A-subunit dictates the interactions of the toxin with blood components.

Hemolytic uremic syndrome (eHUS) is a severe complication of human infections with Shiga toxins (Stxs)-producing Escherichia coli. A key step in the pathogenesis of eHUS is the interaction of Stxs with blood components before the targeting of renal endothelial cells. Here, we show that a single proteolytic cleavage in the Stx2a A-subunit, resulting into two fragments (A1 and A2) linked by a disulfide bridge (cleaved Stx2a), dictates different binding abilities. Uncleaved Stx2a was confirmed to bind to human neutrophils and to trigger leukocyte/platelet aggregate formation, whereas cleaved Stx2a was ineffective. Conversely, binding of complement factor H was confirmed for cleaved Stx2a and not for uncleaved Stx2a. It is worth noting that uncleaved and cleaved Stx2a showed no differences in cytotoxicity for Vero cells or Raji cells, structural conformation, and contaminating endotoxin. These results have been obtained by comparing two Stx2a batches, purified in different laboratories by using different protocols, termed Stx2a(cl; cleaved toxin, Innsbruck) and Stx2a(uncl; uncleaved toxin, Bologna). Stx2a(uncl) behaved as Stx2a(cl) after mild trypsin treatment. In this light, previous controversial results obtained with purified Stx2a has to be critically re-evaluated; furthermore, characterisation of the structure of circulating Stx2a is mandatory to understand eHUS-pathogenesis and to develop therapeutic approaches.

Cancer Site-Specific Multiple microRNA Quantification by Droplet Digital PCR.

Archival formalin-fixed paraffin-embedded (FFPE) tissues represent an extraordinary source of smallRNAs, including microRNAs (miRNAs). Contrary to other RNA molecules, miRNAs are stable, nuclease-resistant and quantifiable even in low quality samples. The accurate assessment of miRNA levels in archival samples is of great interest for many pathological conditions, including cancer. In human tumors, microRNA expression is type-specific and can be used as diagnostic, prognostic or response-to-treatment biomarker. In this study, we provide a method for multiple miRNA quantification in 96-well plates, using EvaGreen-based droplet digital PCR technology and miRCURY LNA miRNA assays. This approach allows the absolute quantification of a customizable panel of miRNAs at the same time and under identical experimental conditions, to be used for diagnostic or prognostic applications.

Human monocytes stimulated by Shiga toxin 1a via globotriaosylceramide release proinflammatory molecules associated with hemolytic uremic syndrome.

The life-threatening sequela of hemorrhagic colitis induced by Shiga toxins (Stx)-producing Escherichia coli (STEC) infections in humans is hemolytic uremic syndrome (HUS), the main cause of acute renal failure in early childhood. The key step in the pathogenesis of HUS is the appearance of Stx in the blood of infected patients because these powerful virulence factors are capable of inducing severe microangiopathic lesions in the kidney. During precocious toxemia, which occurs in patients before the onset of HUS during the intestinal phase, Stx bind to several different circulating cells. An early response of these cells might include the release of proinflammatory mediators associated with the development of HUS. Here, we show that primary human monocytes stimulated with Shiga toxin 1a (Stx1a) through the glycolipid receptor globotriaosylceramide released larger amounts of proinflammatory molecules (IL-1ß, TNFα, IL-6, G-CSF, CXCL8, CCL2, CCL4) than Stx1a-treated neutrophils. The mediators (except IL-1ß) are among the top six proinflammatory mediators found in the sera from patients with HUS in different studies. The molecules appear to be involved in different pathogenetic steps of HUS, i.e. sensitization of renal endothelial cells to the toxin actions (IL-1ß, TNFα), activation of circulating monocytes and neutrophils (CXCL8, CCL2, CCL4) and increase in neutrophil counts in patients with poor prognosis (G-CSF). Hence, a role of circulating monocytes in the very early phases of the pathogenetic process culminating with HUS can be envisaged. Impairment of the events of precocious toxemia would prevent or reduce the risk of HUS in STEC-infected children.

Epigenetic and epitranscriptomic changes in colorectal cancer: Diagnostic, prognostic, and treatment implications.

A cancer cell is the final product of a complex mixture of genetic, epigenetic and epitranscriptomic alterations, whose final interplay contribute to cancer onset and progression. This is specifically true for colorectal cancer, a tumor with a strong epigenetic component, which acts earlier than any other genetic alteration in promoting cancer cell malignant transformation. The pattern of progressive, and usually subtype-specific, DNA and histone modifications that occur in colorectal cancer has been extensively studied in the last decade, providing plenty of data to explore. For this tumor, it became recently evident that also RNA modifications play a relevant role in the activation of oncogenes or repression of tumor suppressor genes. In this review we provide a brief overview of all epigenetic and epitranscriptomic changes that have been found associated to colorectal cancer till now. We explore the impact of these alterations in cancer prognosis and response to treatment and discuss their potential use as cancer biomarkers.

Non-Coding RNAs as Predictive Biomarkers to Current Treatment in Metastatic Colorectal Cancer.

The onset and selection of resistant clones during cancer treatment with chemotherapy or targeted therapy is a major issue in the clinical management of metastatic colorectal cancer patients. It is possible that a more personalized treatment selection, using reliable response-to-therapy predictive biomarkers, could lead to an improvement in the success rate of the proposed therapies. Although the process of biomarker selection and validation could be a long one, requiring solid statistics, large cohorts and multicentric validations, non-coding RNAs (ncRNAs) and in particular microRNAs, proved to be extremely promising in this field. Here we summarize some of the main studies correlating specific ncRNAs with sensitivity/resistance to chemotherapy, anti-VEGF therapy, anti-EGFR therapy and immunotherapy in colorectal cancer (CRC).

Peripheral Inflammatory Markers and Antioxidant Response during the Post-Acute and Chronic Phase after Severe Traumatic Brain Injury.

Traumatic brain injury (TBI) is a mechanical insult to the brain caused by external forces and associated with inflammation and oxidative stress. The patients may show different profiles of neurological recovery and a combination of oxidative damage and inflammatory processes can affect their courses. It is known that an overexpression of cytokines can be seen in peripheral blood in the early hours/days after the injury, but little is known about the weeks and months encompassing the post-acute and chronic phases. In addition, no information is available about the antioxidant responses mediated by the major enzymes that regulate reactive oxygen species levels: superoxide dismutase, catalase, peroxidases, and GSH-related enzymes. This study investigates the 6-month trends of inflammatory markers and antioxidant responses in 22 severe TBI patients with prolonged disorders of consciousness, consecutively recruited in a dedicated neurorehabilitation facility. Patients with a high degree of neurological impairment often show an uncertain outcome. In addition, the profiles of plasma activities were related to the neurological recovery after 12 months. Venous peripheral blood samples were taken blindly as soon as clinical signs and laboratory markers confirmed the absence of infections, 3 and 6 months later. The clinical and neuropsychological assessment continued up to 12 months. Nineteen patients completed the follow-up. In the chronic phase, persistent high plasma levels of cytokines can interfere with cognitive functioning and higher post-acute levels of cytokines [interferon (IFN)-γ, tumor necrosis factor (TNF)-α, IL1b, IL6] are associated with poorer cognitive recoveries 12 months later. Moreover, higher IFN-γ, higher TNF-α, and lower glutathione peroxidase activity are associated with greater disability. The results add evidence of persistent inflammatory response, provide information about long-term imbalance of antioxidant activity, and suggest that the over-production of cytokines and the alteration of the redox homeostasis in the post-acute phase might adversely affect the neurological and functional recovery. Inflammatory and antioxidant activity markers might offer a feasible way to highlight some of the processes opposing recovery after a severe TBI.

Persistent infections, immune-senescence and Alzheimer's disease.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder and the most common cause of dementia. Classical hallmarks of AD such as amyloid deposition and neurofibrillary tangles do not completely explain AD pathogenesis. Recent investigations proposed Aß peptide as an anti-microbial factor. Our previous works suggested that the concomitant presence of single nucleotide polymorphisms (SNPs) from AD genetic studies might impair antiviral defenses and increase the individual susceptibility to herpes virus infection. Viruses of herpes family by inducing frequent cycles of reactivation and latency constantly challenge the immune response and drive the accumulation of memory T cells. However, the immune system is not able to completely eradicate these viruses. The continuous antigen stimulation activates chronic inflammatory responses that may progressively induce neurodegenerative mechanisms in genetically susceptible elderly. The aim of this paper is to suggest new perspectives in clinical pathogenesis of AD with potential prevention and new medical treatment of the age associated cognitive decline.

Peripheral leukocyte expression of the potential biomarker proteins Bdnf, Sirt1, and Psen1 is not regulated by promoter methylation in Alzheimer's disease patients.

The identification of Alzheimer's disease (AD) biomarkers is crucial to support drug discovery. Within putative biomarkers, peripheral Bdnf levels correlate with cognitive decline and AD, although conflicting findings are reported. Sirtuin 1 (Sirt1) serum levels are lower in AD patients and Presenilin 1 (Psen1) is expressed by blood cells. DNA methylation is altered in AD patients, suggesting that epigenetic mechanisms play a role in AD pathophysiology. The objective of this study was to investigate promoter methylation levels of potential biomarkers in AD cases and controls. Peripheral blood DNA methylation levels were analysed by methylation-specific primer real-time PCR. Bdnf promoter methylation levels did not differ between AD patients and controls. Similarly, Sirt1 promoter revealed minimal levels of methylation which did not display significant differences between groups. No significant difference was revealed between AD patients and controls also in Psen1 methylation, showing a large variability of values among subjects. Although peripheral Bdnf expression is associated with differential promoter methylation in psychiatric and neurological disorders, our results suggest that different mechanisms take place in AD. The finding that the control of Sirt1 protein levels in blood is not exerted through the repression of mRNA expression by promoter hypermethylation is in agreement with previous data. In contrast, other studies reported that Psen1 methylation may be increased or decreased in AD patients, suggesting that additional studies are required. In conclusion, this study shows that peripheral levels of the potential AD biomarker proteins Bdnf, Sirt1, and Psen1 are not regulated by different promoter methylation.

Variants in Antiviral Genes are Risk Factors for Cognitive Decline and Dementia.

A gene association study of factors regulating antiviral response such as interferon (IFN)-λ3, also known as IL-28B, mediator complex (Med) 23, and interferon regulatory factor (IRF) 7 with cognitive deterioration and Alzheimer's disease (AD) was performed. Differences in the TT genotype distribution of IL-28B single nucleotide polymorphism (SNP) between AD patients and controls were found. The GG genotype of Med23 gene appeared to influence the progression of the disease, being more frequent in the APOE ɛ4 negative elderly that developed AD during the five year follow-up. Leukocyte positivity for Epstein Barr virus (EBV) and human herpes virus (HHV)-6 DNA was analyzed. Med23 GG genotype correlated with the positivity to HHV-6 DNA. EBV and HHV-6 plasma IgG levels were also investigated and EBV IgG levels were increased in AD with the IRF7 GG genotype. A differential genetic background in genes regulating anti-virus responses was associated with an increased risk of cognitive decline and AD. EBV and HHV-6 appeared to be risk factors for AD in genetically susceptible elderly.

The 21st century epidemic: infections as inductors of neuro-degeneration associated with Alzheimer's Disease.

Alzheimer's disease (AD) is a complex disease resulting in neurodegeneration and cognitive impairment. Investigations on environmental factors implicated in AD are scarce and the etiology of the disease remains up to now obscure. The disease's pathogenesis may be multi-factorial and different etiological factors may converge during aging and induce an activation of brain microglia and macrophages. This microglia priming will result in chronic neuro-inflammation under chronic antigen activation. Infective agents may prime and drive iper-activation of microglia and be partially responsible of the induction of brain inflammation and decline of cognitive performances. Age-associated immune dis-functions induced by chronic sub-clinical infections appear to substantially contribute to the appearance of neuro-inflammation in the elderly. Individual predisposition to less efficient immune responses is another relevant factor contributing to impaired regulation of inflammatory responses and accelerated cognitive decline. Life-long virus infection may play a pivotal role in activating peripheral and central inflammatory responses and in turn contributing to increased cognitive impairment in preclinical and clinical AD.