Progression of Pathogenic Events in Cynomolgus Macaques Infected with Variola Virus

Smallpox, caused by variola virus (VARV), is a devastating human disease that affected millions worldwide until the virus was eradicated in the 1970 s. Subsequent cessation of vaccination has resulted in an immunologically naive human population that would be at risk should VARV be used as an agent of bioterrorism. The development of antivirals and improved vaccines to counter this threat would be facilitated by the development of animal models using authentic VARV. Towards this end, cynomolgus macaques were identified as adequate hosts for VARV, developing ordinary or hemorrhagic smallpox in a dose-dependent fashion. To further refine this model, we performed a serial sampling study on macaques exposed to doses of VARV strain Harper calibrated to induce ordinary or hemorrhagic disease. Several key differences were noted between these models. In the ordinary smallpox model, lymphoid and myeloid hyperplasias were consistently found whereas lymphocytolysis and hematopoietic necrosis developed in hemorrhagic smallpox. Viral antigen accumulation, as assessed immunohistochemically, was mild and transient in the ordinary smallpox model. In contrast, in the hemorrhagic model antigen distribution was widespread and included tissues and cells not involved in the ordinary model. Hemorrhagic smallpox developed only in the presence of secondary bacterial infections – an observation also commonly noted in historical reports of human smallpox. Together, our results support the macaque model as an excellent surrogate for human smallpox in terms of disease onset, acute disease course, and gross and histopathological lesions

Distinct Patterns of DNA Damage Response and Apoptosis Correlate with Jak/Stat and PI3Kinase Response Profiles in Human Acute Myelogenous Leukemia

BACKGROUND:Single cell network profiling (SCNP) utilizing flow cytometry measures alterations in intracellular signaling responses. Here SCNP was used to characterize Acute Myeloid Leukemia (AML) disease subtypes based on survival, DNA damage response and apoptosis pathways. METHODOLOGY AND PRINCIPAL FINDINGS:Thirty four diagnostic non-M3 AML samples from patients with known clinical outcome were treated with a panel of myeloid growth factors and cytokines, as well as with apoptosis-inducing agents. Analysis of induced Jak/Stat and PI3K pathway responses in blasts from individual patient samples identified subgroups with distinct signaling profiles that were not seen in the absence of a modulator. In vitro exposure of patient samples to etoposide, a DNA damaging agent, revealed three distinct "DNA damage response (DDR)/apoptosis" profiles: 1) AML blasts with a defective DDR and failure to undergo apoptosis; 2) AML blasts with proficient DDR and failure to undergo apoptosis; 3) AML blasts with proficiency in both DDR and apoptosis pathways. Notably, AML samples from clinical responders fell within the "DDR/apoptosis" proficient profile and, as well, had low PI3K and Jak/Stat signaling responses. In contrast, samples from clinical non responders had variable signaling profiles often with in vitro apoptotic failure and elevated PI3K pathway activity. Individual patient samples often harbored multiple, distinct, leukemia-associated cell populations identifiable by their surface marker expression, functional performance of signaling pathway in the face of cytokine or growth factor stimulation, as well as their response to apoptosis-inducing agents. CONCLUSIONS AND SIGNIFICANCE:Characterizing and tracking changes in intracellular pathway profiles in cell subpopulations both at baseline and under therapeutic pressure will likely have important clinical applications, potentially informing the selection of beneficial targeted agents, used either alone or in combination with chemotherapy

Cluster analysis of behavioural and event-related potentials during a contingent negative variation paradigm in remitting-relapsing and benign forms of multiple sclerosis

<p>Abstract</p> <p>Background</p> <p>Event-related potentials (ERPs) may be used as a highly sensitive way of detecting subtle degrees of cognitive dysfunction. On the other hand, impairment of cognitive skills is increasingly recognised as a hallmark of patients suffering from multiple sclerosis (MS). We sought to determine the psychophysiological pattern of information processing among MS patients with the relapsing-remitting form of the disease and low physical disability considered as two subtypes: 'typical relapsing-remitting' (RRMS) and 'benign MS' (BMS). Furthermore, we subjected our data to a cluster analysis to determine whether MS patients and healthy controls could be differentiated in terms of their psychophysiological profile.</p> <p>Methods</p> <p>We investigated MS patients with RRMS and BMS subtypes using event-related potentials (ERPs) acquired in the context of a Posner visual-spatial cueing paradigm. Specifically, our study aimed to assess ERP brain activity in response preparation (contingent negative variation -CNV) and stimuli processing in MS patients. Latency and amplitude of different ERP components (P1, eN1, N1, P2, N2, P3 and late negativity -LN) as well as behavioural responses (reaction time -RT; correct responses -CRs; and number of errors) were analyzed and then subjected to cluster analysis.</p> <p>Results</p> <p>Both MS groups showed delayed behavioural responses and enhanced latency for long-latency ERP components (P2, N2, P3) as well as relatively preserved ERP amplitude, but BMS patients obtained more important performance deficits (lower CRs and higher RTs) and abnormalities related to the latency (N1, P3) and amplitude of ERPs (eCNV, eN1, LN). However, RRMS patients also demonstrated abnormally high amplitudes related to the preparation performance period of CNV (cCNV) and post-processing phase (LN). Cluster analyses revealed that RRMS patients appear to make up a relatively homogeneous group with moderate deficits mainly related to ERP latencies, whereas BMS patients appear to make up a rather more heterogeneous group with more severe information processing and attentional deficits.</p> <p>Conclusions</p> <p>Our findings are suggestive of a slowing of information processing for MS patients that may be a consequence of demyelination and axonal degeneration, which also seems to occur in MS patients that show little or no progression in the physical severity of the disease over time.</p

Decreasing the expression of PICALM reduces endocytosis and the activity of β-secretase: Implications for Alzheimer's disease

© 2016 The Author(s). Background: Polymorphisms in the gene for phosphatidylinositol binding clathrin assembly protein (PICALM), an endocytic-related protein, are associated with a small, increased risk of developing Alzheimer's disease (AD), strongly suggesting that changes in endocytosis are involved in the aetiology of the disease. We have investigated the involvement of PICALM in the processing of amyloid precursor protein (APP) to understand how PICALM could be linked to the development of AD. We used siRNA to deplete levels of PICALM, its isoforms and clathrin heavy chain in the human brain-derived H4 neuroglioma cell line that expresses endogenous levels of APP. We then used Western blotting, ELISA and immunohistochemistry to detect intra- and extracellular protein levels of endocytic-related proteins, APP and APP metabolites including β-amyloid (Aβ). Levels of functional endocytosis were quantified using ALEXA 488-conjugated transferrin and flow cytometry as a marker of clathrin-mediated endocytosis (CME). Results: Following depletion of all the isoforms of PICALM by siRNA in H4 cells, levels of intracellular APP, intracellular β-C-terminal fragment (β-CTF) and secreted sAPPβ (APP fragments produced by β-secretase cleavage) were significantly reduced but Aβ40 was not affected. Functional endocytosis was significantly reduced after both PICALM and clathrin depletion, highlighting the importance of PICALM in this process. However, depletion of clathrin did not affect APP but did reduce β-CTF levels. PICALM depletion altered the intracellular distribution of clathrin while clathrin reduction affected the subcellular pattern of PICALM labelling. Both PICALM and clathrin depletion reduced the expression of BACE1 mRNA and PICALM siRNA reduced protein levels. Individual depletion of PICALM isoforms 1 and 2 did not affect APP levels while clathrin depletion had a differential effect on the isoforms, increasing isoform 1 while decreasing isoform 2 expression. Conclusions: The depletion of PICALM in brain-derived cells has significant effects on the processing of APP, probably by reducing CME. In particular, it affects the production of β-CTF which is increasingly considered to be an important mediator in AD independent of Aβ. Thus a decrease in PICALM expression in the brain could be beneficial to slow or prevent the development of AD

Prebiotic Effects of Wheat Arabinoxylan Related to the Increase in Bifidobacteria, Roseburia and Bacteroides/Prevotella in Diet-Induced Obese Mice

BACKGROUND: Alterations in the composition of gut microbiota--known as dysbiosis--has been proposed to contribute to the development of obesity, thereby supporting the potential interest of nutrients targeting the gut with beneficial effect for host adiposity. We test the ability of a specific concentrate of water-extractable high molecular weight arabinoxylans (AX) from wheat to modulate both the gut microbiota and lipid metabolism in high-fat (HF) diet-induced obese mice. METHODOLOGY/PRINCIPAL FINDINGS: Mice were fed either a control diet (CT) or a HF diet, or a HF diet supplemented with AX (10% w/w) during 4 weeks. AX supplementation restored the number of bacteria that were decreased upon HF feeding, i.e. Bacteroides-Prevotella spp. and Roseburia spp. Importantly, AX treatment markedly increased caecal bifidobacteria content, in particular Bifidobacterium animalis lactis. This effect was accompanied by improvement of gut barrier function and by a lower circulating inflammatory marker. Interestingly, rumenic acid (C18:2 c9,t11) was increased in white adipose tissue due to AX treatment, suggesting the influence of gut bacterial metabolism on host tissue. In parallel, AX treatment decreased adipocyte size and HF diet-induced expression of genes mediating differentiation, fatty acid uptake, fatty acid oxidation and inflammation, and decreased a key lipogenic enzyme activity in the subcutaneous adipose tissue. Furthermore, AX treatment significantly decreased HF-induced adiposity, body weight gain, serum and hepatic cholesterol accumulation and insulin resistance. Correlation analysis reveals that Roseburia spp. and Bacteroides/Prevotella levels inversely correlate with these host metabolic parameters. CONCLUSIONS/SIGNIFICANCE: Supplementation of a concentrate of water-extractable high molecular weight AX in the diet counteracted HF-induced gut dysbiosis together with an improvement of obesity and lipid-lowering effects. We postulate that hypocholesterolemic, anti-inflammatory and anti-obesity effects are related to changes in gut microbiota. These data support a role for wheat AX as interesting nutrients with prebiotic properties related to obesity prevention

Finding New Genes for Non-Syndromic Hearing Loss through an In Silico Prioritization Study

At present, 51 genes are already known to be responsible for Non-Syndromic hereditary Hearing Loss (NSHL), but the knowledge of 121 NSHL-linked chromosomal regions brings to the hypothesis that a number of disease genes have still to be uncovered. To help scientists to find new NSHL genes, we built a gene-scoring system, integrating Gene Ontology, NCBI Gene and Map Viewer databases, which prioritizes the candidate genes according to their probability to cause NSHL. We defined a set of candidates and measured their functional similarity with respect to the disease gene set, computing a score () that relies on the assumption that functionally related genes might contribute to the same (disease) phenotype. A Kolmogorov-Smirnov test, comparing the pair-wise distribution on the disease gene set with the distribution on the remaining human genes, provided a statistical assessment of this assumption. We found at a p-value that the former pair-wise is greater than the latter, justifying a prioritization strategy based on the functional similarity of candidate genes respect to the disease gene set. A cross-validation test measured to what extent the ranking for NSHL is different from a random ordering: adding 15% of the disease genes to the candidate gene set, the ranking of the disease genes in the first eight positions resulted statistically different from a hypergeometric distribution with a p-value and a power. The twenty top-scored genes were finally examined to evaluate their possible involvement in NSHL. We found that half of them are known to be expressed in human inner ear or cochlea and are mainly involved in remodeling and organization of actin formation and maintenance of the cilia and the endocochlear potential. These findings strongly indicate that our metric was able to suggest excellent NSHL candidates to be screened in patients and controls for causative mutations

Antiinflammatory Therapy with Canakinumab for Atherosclerotic Disease

Background: Experimental and clinical data suggest that reducing inflammation without affecting lipid levels may reduce the risk of cardiovascular disease. Yet, the inflammatory hypothesis of atherothrombosis has remained unproved. Methods: We conducted a randomized, double-blind trial of canakinumab, a therapeutic monoclonal antibody targeting interleukin-1β, involving 10,061 patients with previous myocardial infarction and a high-sensitivity C-reactive protein level of 2 mg or more per liter. The trial compared three doses of canakinumab (50 mg, 150 mg, and 300 mg, administered subcutaneously every 3 months) with placebo. The primary efficacy end point was nonfatal myocardial infarction, nonfatal stroke, or cardiovascular death. RESULTS: At 48 months, the median reduction from baseline in the high-sensitivity C-reactive protein level was 26 percentage points greater in the group that received the 50-mg dose of canakinumab, 37 percentage points greater in the 150-mg group, and 41 percentage points greater in the 300-mg group than in the placebo group. Canakinumab did not reduce lipid levels from baseline. At a median follow-up of 3.7 years, the incidence rate for the primary end point was 4.50 events per 100 person-years in the placebo group, 4.11 events per 100 person-years in the 50-mg group, 3.86 events per 100 person-years in the 150-mg group, and 3.90 events per 100 person-years in the 300-mg group. The hazard ratios as compared with placebo were as follows: in the 50-mg group, 0.93 (95% confidence interval [CI], 0.80 to 1.07; P = 0.30); in the 150-mg group, 0.85 (95% CI, 0.74 to 0.98; P = 0.021); and in the 300-mg group, 0.86 (95% CI, 0.75 to 0.99; P = 0.031). The 150-mg dose, but not the other doses, met the prespecified multiplicity-adjusted threshold for statistical significance for the primary end point and the secondary end point that additionally included hospitalization for unstable angina that led to urgent revascularization (hazard ratio vs. placebo, 0.83; 95% CI, 0.73 to 0.95; P = 0.005). Canakinumab was associated with a higher incidence of fatal infection than was placebo. There was no significant difference in all-cause mortality (hazard ratio for all canakinumab doses vs. placebo, 0.94; 95% CI, 0.83 to 1.06; P = 0.31). Conclusions: Antiinflammatory therapy targeting the interleukin-1β innate immunity pathway with canakinumab at a dose of 150 mg every 3 months led to a significantly lower rate of recurrent cardiovascular events than placebo, independent of lipid-level lowering. (Funded by Novartis; CANTOS ClinicalTrials.gov number, NCT01327846.