TLR3 controls constitutive IFN-β antiviral immunity in human fibroblasts and cortical neurons

Human herpes simplex virus 1 (HSV-1) encephalitis can be caused by inborn errors of the TLR3 pathway, resulting in impairment of CNS cell-intrinsic antiviral immunity. Deficiencies of the TLR3 pathway impair cell-intrinsic immunity to vesicular stomatitis virus (VSV) and HSV-1 in fibroblasts, and to HSV-1 in cortical but not trigeminal neurons. The underlying molecular mechanism is thought to involve impaired IFN-α/β induction by the TLR3 recognition of dsRNA viral intermediates or by-products. However, we show here that human TLR3 controls constitutive levels of IFNB mRNA and secreted bioactive IFN-β protein, and thereby also controls constitutive mRNA levels for IFN-stimulated genes (ISGs) in fibroblasts. Tlr3^–/– mouse embryonic fibroblasts also have lower basal ISG levels. Moreover, human TLR3 controls basal levels of IFN-β secretion and ISG mRNA in induced pluripotent stem cell–derived cortical neurons. Consistently, TLR3-deficient human fibroblasts and cortical neurons are vulnerable not only to both VSV and HSV-1, but also to several other families of viruses. The mechanism by which TLR3 restricts viral growth in human fibroblasts and cortical neurons in vitro and, by inference, by which the human CNS prevents infection by HSV-1 in vivo, is therefore based on the control of early viral infection by basal IFN-β immunity.     

Amyloid-β oligomer synaptotoxicity is mimicked by oligomers of the model protein HypF-N

Protein misfolded oligomers are thought to be the primary pathogenic species in many protein deposition diseases. Oligomers by the amyloid-β peptide play a central role in Alzheimer's disease pathogenesis, being implicated in synaptic dysfunction. Here we show that the oligomers formed by a protein that has no link with human disease, namely the N-terminal domain of HypF from Escherichia coli (HypF-N), are also synaptotoxic. HypF-N oligomers were found to (i) colocalize with post-synaptic densities in primary rat hippocampal neurons; (ii) induce impairment of long-term potentiation in rat hippocampal slices; and (iii) impair spatial learning of rats in the Morris Water Maze test. By contrast, the native protein and control nontoxic oligomers had none of such effects. These results raise the importance of using HypF-N oligomers as a valid tool to investigate the pathogenesis of Alzheimer's disease, with advantages over other systems for their stability, reproducibility, and costs. The results also suggest that, in the context of a compromised protein homeostasis resulting from aggregation of the amyloid β peptide, a number of oligomeric species sharing common synaptotoxic activity can arise and cooperate in the pathogenesis of the disease.     

EphA2-mediated mesenchymal–amoeboid transition induced by endothelial progenitor cells enhances metastatic spread due to cancer-associated fibroblasts

Tumor progression is deeply influenced by epigenetic changes induced by tumor stroma. Cancer-associated fibroblasts (CAFs) have been reported to promote epithelial–mesenchymal transition in cancer cells, thereby enhancing their aggressiveness and stem-like properties. As CAFs are able to recruit endothelial progenitor cells (EPCs) to tumor site, we aim to investigate their interplay for prostate carcinoma progression. Both prostate CAFs and cancer cells actively recruit EPCs, known to affect tumor progression through increased vasculogenesis. EPCs synergize with CAFs to further promote epigenetic plasticity of cancer cells, through a mesenchymal-to-amoeboid transition. Indeed, after fibroblasts have engaged epithelial–mesenchymal transition in cancer cells, a further shift towards amoeboid motility is promoted by EPCs through contact-mediated triggering of the bidirectional ephrinA1/EphA2 signaling. The activation of ephrinA1 reverse pathway enhances EPC-induced neo-vascularization, thus promoting tumor growth, while EphA2 forward signaling elicits mesenchymal–amoeboid transition in cancer cells, favoring their adhesion to endothelium, transendothelial migration, and lung metastatic colonization. We therefore underscore that the metastatic advantage given by tumor microenvironment embraces different motility strategies and propose EphA2-targeted tools as useful adjuvants in anti-metastatic treatments.     

Characterization of Neuroendocrine Tumors of the Pancreas by Real-Time Quantitative Polymerase Chain Reaction. A Methodological Approach

The aim of this study was to assess the suitability of using real-time quantitative PCR (RT-qPCR) to characterize neuroendocrine (NE) tumors of the pancreas. For a series of tumors, we evaluated several genes of interest, and the data were matched with the “classical” immunohistochemical (IHC) features. In 21 cases, we extracted RNA from formalin-fixed paraffin-embedded (FFPE) blocks, and in nine cases, we also extracted RNA from fresh-frozen tissue. The RT-qPCR procedure was performed using two sets of customized arrays. The test using the first set, covering 96 genes of interest, was focused on assessing the feasibility of the procedure, and the results were used to select 18 genes indicative of NE differentiation, clinical behavior, and therapeutic responsiveness for use in the second set of arrays. Threshold cycle (Ct) values were used to calculate the fold-changes in gene expression using the 2-??Ct method. Statistical procedures were used to analyze the results, which were matched with the IHC and follow-up data. Material from fresh-frozen samples performed better in terms of the level of amplification, but acceptable and concordant results were also obtained from FFPE samples. In addition, high concordance was observed between the mRNA and protein expression levels of somatostatin receptor type 2A (R?=?0.52, p?=?0.016). Genes associated with NE differentiation, as well as the gastrin-releasing peptide receptor and O-6-methylguanine-DNA methyltransferase genes, were underexpressed, whereas angiogenesis-associated markers (CDH13 and SLIT2) were overexpressed in tissues with malignant behavior. The RT-qPCR procedure is practical and feasible in economic terms for the characterization of NE tumors of the pancreas and can complement morphological and IHC-based evaluations. Thus, the results of the RT-qPCR procedure might offer an objective basis for therapeutic choices.     

Neurological examination findings to predict limitations in mobility and falls in older persons without a history of neurological disease

PURPOSE: To estimate the prevalence of neurological signs and their association with limitations in mobility and falls in a sample of older persons without known neurological disease. METHODS: A neurologist examined 818 participants from the InCHIANTI study who were aged > or =65 years and who did not have cognitive impairment, treatment with neuroleptics, and a history of neurological disease. Mobility was assessed as walking speed and self-reported ability to walk at least 1 km without difficulty. Participants were asked to report falls that had occurred in the previous 12 months. RESULTS: Less than 20% (160/818) of participants had no neurological signs. Neurological signs were more prevalent in older participants and those with impaired mobility. When all neurological signs were included in sex-and age-adjusted multivariate models, 10 were mutually independent correlates of poor mobility. After adjusting for age and sex, the number of neurological signs was associated with progressively slower walking speed (P <0.001), a higher probability of reported inability to walk 1 km (P <0.001), and a history of falls (P <0.05). CONCLUSION: Neurological signs are independent correlates of limitations in mobility and falls in older persons who have no clear history of neurological disease.     

Manganese superoxide dismutase activity and incidence of hepatocellular carcinoma in patients with Child-Pugh class A liver cirrhosis: a 7-year follow-up study.

AIMS: To evaluate possible modifications in the manganese superoxide dismutase (MnSOD) activity during neoplastic transformation of a cirrhotic liver and to find out whether its assessment may have predictive value to identify cirrhotic patients at a higher risk of hepatocellular carcinoma (HCC). METHODS: Seventy-one consecutive subjects with Child-Pugh class A liver cirrhosis were recruited. At the time of enrolment, HCC was diagnosed in 20 cirrhotic patients. The 51 cirrhotic patients without HCC were followed up for the occurrence of tumour by 6-monthly screening for 7 years. During follow-up, 16 patients developed HCC. Seventy healthy subjects formed the control group. MnSOD activity was assayed spectrophotometrically. RESULTS: Serum MnSOD activity was significantly lower in 70 healthy subjects compared with 51 cirrhotic patients and 20 cirrhotic patients with HCC. Cirrhotic patients who developed HCC during follow-up showed significantly higher values of MnSOD activity than HCC-free patients. The best cut-off of MnSOD activity was 0.40 U/ml. At this cut-off, chi2 analysis revealed that MnSOD activity was significantly different between the HCC-free cirrhotic patients and cirrhotic patients who developed HCC. CONCLUSION: The present findings suggest that during neoplastic transformation of cirrhotic liver, an increase in MnSOD activity may occur already during the precancerous phase, making this enzyme a probable malignancy-associated parameter.     

Usefulness of chronotropic incompetence to dipyridamole in predicting myocardial perfusion defects in heart transplant recipients

The aim of this report was to assess the relation between heart rate response to dipyridamole infusion and perfusion defects at quantitative sestamibi single-photon emission computed tomographic imaging. We demonstrated in 166 heart transplant recipients that chronotropic incompetence to dipyridamole is the only significant and independent predictor of perfusion defects.