Sensitive detection of chromatin coassociations using enhanced chromosome conformation capture on chip

Chromosome conformation capture (3C) is a powerful technique for analyzing spatial chromatin organization in vivo. Technical variants of the assay ('4C') allow the systematic detection of genome-wide coassociations with bait sequences of interest, enabling the nuclear environments of specific genes to be probed. We describe enhanced 4C (e4C, enhanced chromosome conformation capture on chip), a technique incorporating additional enrichment steps for bait-specific sequences, and thus improving sensitivity in the detection of weaker, distal chromatin coassociations. In brief, e4C entails the fixation, restriction digestion and ligation steps of conventional 3C, with an optional chromatin immunoprecipitation (ChIP) step to select for subsets of chromatin coassociations, followed by bait enrichment by biotinylated primer extension and pull-down, adapter ligation and PCR amplification. Chromatin coassociations with the bait sequence can then be assessed by hybridizing e4C products to microarrays or sequencing. The e4C procedure takes approximately 1 week to go from tissue to DNA ready for microarray hybridization.     

Synchronous versus asynchronous oscillations for antigenically varying Plasmodium falciparum with host immune response

We consider a deterministic intra-host model for Plasmodium falciparum (Pf) malaria infection, which accounts for antigenic variation between n clonal variants of PfEMP1 and the corresponding host immune response (IR). Specifically, the model separates the IR into two components, specific and cross-reactive, respectively, in order to demonstrate that the latter can be a mechanism for the sequential appearance of variants observed in actual Pf infections. We show that a strong variant-specific IR relative to the cross-reactive IR favours the asynchronous oscillations (sequential dominance) over the synchronous oscillations in a number of ways. The decay rate of asynchronous oscillations is smaller than that for the synchronous oscillations, allowing for the parasite to survive longer. With the introduction of a delay in the stimulation of the IR, we show that only a small delay is necessary to cause persistent asynchronous oscillations and that a strong variant-specific IR increases the amplitude of the asynchronous oscillations.     

Measurement of Phospholipids May Improve Diagnostic Accuracy in Ovarian Cancer

Background

More than two-thirds of women who undergo surgery for suspected ovarian neoplasm do not have cancer. Our previous results suggest phospholipids as potential biomarkers of ovarian cancer. In this study, we measured the serum levels of multiple phospholipids among women undergoing surgery for suspected ovarian cancer to identify biomarkers that better predict whether an ovarian mass is malignant.

Methodology/Principal Findings

We obtained serum samples preoperatively from women with suspected ovarian cancer enrolled through a prospective, population-based rapid ascertainment system. Samples were analyzed from all women in whom a diagnosis of epithelial ovarian cancer (EOC) was confirmed and from benign disease cases randomly selected from the remaining (non-EOC) samples. We measured biologically relevant phospholipids using liquid chromatography/electrospray ionization mass spectrometry. We applied a powerful statistical and machine learning approach, Hybrid huberized support vector machine (HH-SVM) to prioritize phospholipids to enter the biomarker models, and used cross-validation to obtain conservative estimates of classification error rates.

Results

The HH-SVM model using the measurements of specific combinations of phospholipids supplements clinical CA125 measurement and improves diagnostic accuracy. Specifically, the measurement of phospholipids improved sensitivity (identification of cases with preoperative CA125 levels below 35) among two types of cases in which CA125 performance is historically poor - early stage cases and those of mucinous histology. Measurement of phospholipids improved the identification of early stage cases from 65% (based on CA125) to 82%, and mucinous cases from 44% to 88%.

Conclusions/Significance

Levels of specific serum phospholipids differ between women with ovarian cancer and those with benign conditions. If validated by independent studies in the future, these biomarkers may serve as an adjunct at the time of clinical presentation, to distinguish between women with ovarian cancer and those with benign conditions with shared symptoms and features.     

Chemical Alarm Cues Are Conserved within the Coral Reef Fish Family Pomacentridae

Fishes are known to use chemical alarm cues from both conspecifics and heterospecifics to assess local predation risks and enhance predator detection. Yet it is unknown how recognition of heterospecific cues arises for coral reef fishes. Here, we test if naïve juvenile fish have an innate recognition of heterospecific alarm cues. We also examine if there is a relationship between the intensity of the antipredator response to these cues and the degree to which species are related to each other. Naïve juvenile anemone fish, Amphiprion percula, were tested to see if they displayed antipredator responses to chemical alarm cues from four closely related heterospecific species (family Pomacentridae), a distantly related sympatric species (Asterropteryx semipunctatus) and a saltwater (control). Juveniles displayed significant reductions in foraging rate when exposed to all four confamilial heterospecific species but they did not respond to the distantly related sympatric species or the saltwater control. There was also a strong relationship between the intensity of the antipredator response and the extent to which species were related, with responses weakening as species became more distantly related. These findings demonstrate that chemical alarm cues are conserved within the pomacentrid family, providing juveniles with an innate recognition of heterospecific alarm cues as predicted by the phylogenetic relatedness hypothesis.     

Activation of invariant NKT cells ameliorates experimental ocular autoimmunity by a mechanism involving innate IFN-gamma production and dampening of the adaptive Th1 and Th17 responses

Invariant NKT cells (iNKT cells) have been reported to play a role not only in innate immunity but also to regulate several models of autoimmunity. Furthermore, iNKT cells are necessary for the generation of the prototypic eye-related immune regulatory phenomenon, anterior chamber associated immune deviation (ACAID). In this study, we explore the role of iNKT cells in regulation of autoimmunity to retina, using a model of experimental autoimmune uveitis (EAU) in mice immunized with a uveitogenic regimen of the retinal Ag, interphotoreceptor retinoid-binding protein. Natural strain-specific variation in iNKT number or induced genetic deficiencies in iNKT did not alter baseline susceptibility to EAU. However, iNKT function seemed to correlate with susceptibility and its pharmacological enhancement in vivo by treatment with iNKT TCR ligands at the time of uveitogenic immunization reproducibly ameliorated disease scores. Use of different iNKT TCR ligands revealed dependence on the elicited cytokine profile. Surprisingly, superior protection against EAU was achieved with alpha-C-GalCer, which induces a strong IFN-gamma but only a weak IL-4 production by iNKT cells, in contrast to the ligands alpha-GalCer (both IFN-gamma and IL-4) and OCH (primarily IL-4). The protective effect of alpha-C-GalCer was associated with a reduction of adaptive Ag-specific IFN-gamma and IL-17 production and was negated by systemic neutralization of IFN-gamma. These data suggest that pharmacological activation of iNKT cells protects from EAU at least in part by a mechanism involving innate production of IFN-gamma and a consequent dampening of the Th1 as well as the Th17 effector responses.     

Dipeptidyl peptidase I-dependent neutrophil recruitment modulates the inflammatory response to Sendai virus infection

The role of innate immunity in the pathogenesis of asthma is unclear. Although increased presence of neutrophils is associated with persistent asthma and asthma exacerbations, how neutrophils participate in the pathogenesis of asthma remains controversial. In this study, we show that the absence of dipeptidyl peptidase I (DPPI), a lysosomal cysteine protease found in neutrophils, dampens the acute inflammatory response and the subsequent mucous cell metaplasia that accompanies the asthma phenotype induced by Sendai virus infection. This attenuated phenotype is accompanied by a significant decrease in the accumulation of neutrophils and the local production of CXCL2, TNF, IL-1beta, and IL-6 in the lung of infected DPPI-/- mice. Adoptive transfer of DPPI-sufficient neutrophils into DPPI-/- mice restored the levels of CXCL2 and enhanced cytokine production on day 4 postinfection and subsequent mucous cell metaplasia on day 21 postinfection. These results indicate that DPPI and neutrophils play a critical role in Sendai virus-induced asthma phenotype as a result of a DPPI-dependent neutrophil recruitment and cytokine response.     

Block of Persistent Late Na+ Currents by Antidepressant Sertraline and Paroxetine

Antidepressants, such as traditional tricyclic antidepressants (TCAs), are the first-line treatment for various pain syndromes. Available evidence indicates that TCAs may target Na⁺ channels for their analgesic action. In this report, we examined the effects of contemporary antidepressants sertraline and paroxetine on (1) neuronal Na⁺ channels expressed in GH₃ cells and (2) muscle rNav1.4 Na⁺ channels heterologously expressed in Hek293t cells. Our results showed that both antidepressants blocked Na⁺ channels in a highly state-dependent manner. The 50% inhibitory concentrations (IC₅₀) for sertraline and paroxetine ranged ∼18–28 μm for resting block and ∼2–8 μm for inactivated block of neuronal and rNav1.4 Na⁺ channels. Surprisingly, the IC₅₀ values for both drugs were about 0.6–0.7 μm for the open channel block of persistent late Na⁺ currents generated through inactivation-deficient rNav1.4 mutant Na⁺ channels. For comparison, the open channel block in neuronal hNav1.7 counterparts yielded IC₅₀ values around 0.3–0.4 μm for both drugs. Receptor mapping using fast inactivation-deficient rNav1.4-F1579A/K mutants with reduced affinities toward local anesthetics (LAs) and TCAs indicated that the F1579 residue is not involved in the binding of sertraline and paroxetine. Thus, sertraline and paroxetine are potent open channel blockers that target persistent late Na⁺ currents preferentially, but their block is not mediated via the phenylalanine residue at the known LA/TCA receptor site.     

Spontaneous colitis occurrence in transgenic mice with altered B7-mediated costimulation

The B7 costimulatory molecules govern many aspects of T cell immune responses by interacting with CD28 for costimulation, but also with CTLA-4 for immune suppression. Although blockade of CTLA-4 with Ab in humans undergoing cancer immune therapy has led to some cases of inflammatory bowel disease, spontaneous animal models of colitis that depend upon modulation of B7 interactions have not been previously described. In this study, we demonstrate that mice expressing a soluble B7-2 Ig Fc chimeric protein spontaneously develop colitis that is dependent on CD28-mediated costimulation of CD4(+) T cells. We show that the chimeric protein has mixed agonistic/antagonist properties, and that it acts in part by blocking the cell intrinsic effects on T cell activation of engagement of CTLA-4. Disease occurred in transgenic mice that lack expression of the endogenous B7 molecules (B7 double knock-out mice), because of the relatively weak costimulatory delivered by the chimeric protein. Surprisingly, colitis was more severe in this context, which was associated with the decreased number of Foxp3(+) regulatory T cells in transgenic B7 double knock-out mice. This model provides an important tool for examining how B7 molecules and their effects on CTLA-4 modulate T cell function and the development of inflammatory diseases.     

Cutting edge: activation by innate cytokines or microbial antigens can cause arrest of natural killer T cell patrolling of liver sinusoids

Natural killer T (NKT) cells are innate-like lymphocytes that rapidly secrete large amounts of effector cytokines upon activation. Recognition of alpha-linked glycolipids presented by CD1d leads to the production of IL-4, IFN-gamma, or both, while direct activation by the synergistic action of IL-12 and IL-18 leads to IFN-gamma production only. We previously reported that in vitro cultured dendritic cells can modulate NKT cell activation and, using intravital fluorescence laser scanning microscopy, we reported that the potent stimulation of NKT cells results in arrest within hepatic sinusoids. In this study, we examine the relationship between murine NKT cell patrolling and activation. We report that NKT cell arrest results from activation driven by limiting doses of a bacteria-derived weak agonist, galacturonic acid-containing glycosphingolipid, or a synthetic agonist, alpha-galactosyl ceramide. Interestingly, NKT cell arrest also results from IL-12 and IL-18 synergistic activation. Thus, innate cytokines and natural microbial TCR agonists trigger sinusoidal NKT cell arrest and an effector response.