Spatial heterogeneity and peptide availability determine CTL killing efficiency in vivo

The rate at which a cytotoxic T lymphocyte (CTL) can survey for infected cells is a key ingredient of models of vertebrate immune responses to intracellular pathogens. Estimates have been obtained using in vivo cytotoxicity assays in which peptide-pulsed splenocytes are killed by CTL in the spleens of immunised mice. However the spleen is a heterogeneous environment and splenocytes comprise multiple cell types. Are some cell types intrinsically more susceptible to lysis than others? Quantitatively, what impacts are made by the spatial distribution of targets and effectors, and the level of peptide-MHC on the target cell surface? To address these questions we revisited the splenocyte killing assay, using CTL specific for an epitope of influenza virus. We found that at the cell population level T cell targets were killed more rapidly than B cells. Using modeling, quantitative imaging and in vitro killing assays we conclude that this difference in vivo likely reflects different migratory patterns of targets within the spleen and a heterogeneous distribution of CTL, with no detectable difference in the intrinsic susceptibilities of the two populations to lysis. Modeling of the stages involved in the detection and killing of peptide-pulsed targets in vitro revealed that peptide dose influenced the ability of CTL to form conjugates with targets but had no detectable effect on the probability that conjugation resulted in lysis, and that T cell targets took longer to lyse than B cells. We also infer that incomplete killing in vivo of cells pulsed with low doses of peptide may be due to a combination of heterogeneity in peptide uptake and the dissociation, but not internalisation, of peptide-MHC complexes. Our analyses demonstrate how population-averaged parameters in models of immune responses can be dissected to account for both spatial and cellular heterogeneity

Dynamic Imaging of CD8+ T Cells and Dendritic Cells during Infection with Toxoplasma gondii

To better understand the initiation of CD8+ T cell responses during infection, the primary response to the intracellular parasite Toxoplasma gondii was characterized using 2-photon microscopy combined with an experimental system that allowed visualization of dendritic cells (DCs) and parasite specific CD8+ T cells. Infection with T. gondii induced localization of both these populations to the sub-capsular/interfollicular region of the draining lymph node and DCs were required for the expansion of the T cells. Consistent with current models, in the presence of cognate antigen, the average velocity of CD8+ T cells decreased. Unexpectedly, infection also resulted in modulation of the behavior of non-parasite specific T cells. This TCR-independent process correlated with the re-modeling of the lymph node micro-architecture and changes in expression of CCL21 and CCL3. Infection also resulted in sustained interactions between the DCs and CD8+ T cells that were visualized only in the presence of cognate antigen and were limited to an early phase in the response. Infected DCs were rare within the lymph node during this time frame; however, DCs presenting the cognate antigen were detected. Together, these data provide novel insights into the earliest interaction between DCs and CD8+ T cells and suggest that cross presentation by bystander DCs rather than infected DCs is an important route of antigen presentation during toxoplasmosis

Epitope Density Influences CD8+ Memory T Cell Differentiation

The generation of long-lived memory T cells is critical for successful vaccination but the factors controlling their differentiation are still poorly defined. We tested the hypothesis that the strength of T cell receptor (TCR) signaling contributed to memory CD8(+) T cell generation.We manipulated the density of antigenic epitope presented by dendritic cells to mouse naïve CD8(+) T cells, without varying TCR affinity. Our results show that a two-fold decrease in antigen dose selectively affects memory CD8(+) T cell generation without influencing T cell expansion and acquisition of effector functions. Moreover, we show that low antigen dose alters the duration of the interaction between T cells and dendritic cells and finely tunes the expression level of the transcription factors Eomes and Bcl6. Furthermore, we demonstrate that priming with higher epitope density results in a 2-fold decrease in the expression of Neuron-derived orphan nuclear receptor 1 (Nor-1) and this correlates with a lower level of conversion of Bcl-2 into a pro-apoptotic molecule and an increased number of memory T cells.Our results show that the amount of antigen encountered by naïve CD8(+) T cells following immunization with dendritic cells does not influence the generation of functional effector CD8(+) T cells but rather the number of CD8(+) memory T cells that persist in the host. Our data support a model where antigenic epitope density sensed by CD8(+) T cells at priming influences memory generation by modulating Bcl6, Eomes and Nor-1 expression

Nanopore-based kinetics analysis of individual antibody-channel and antibody-antigen interactions

<p>Abstract</p> <p>Background</p> <p>The UNO/RIC Nanopore Detector provides a new way to study the binding and conformational changes of individual antibodies. Many critical questions regarding antibody function are still unresolved, questions that can be approached in a new way with the nanopore detector.</p> <p>Results</p> <p>We present evidence that different forms of channel blockade can be associated with the same antibody, we associate these different blockades with different orientations of "capture" of an antibody in the detector's nanometer-scale channel. We directly detect the presence of antibodies via reductions in channel current. Changes to blockade patterns upon addition of antigen suggest indirect detection of antibody/antigen binding. Similarly, DNA-hairpin anchored antibodies have been studied, where the DNA linkage is to the carboxy-terminus at the base of the antibody's Fc region, with significantly fewer types of (lengthy) capture blockades than was observed for free (un-bound) IgG antibody. The introduction of chaotropic agents and its effects on protein-protein interactions have also been observed.</p> <p>Conclusion</p> <p>Nanopore-based approaches may eventually provide a direct analysis of the complex conformational "negotiations" that occur upon binding between proteins.</p

RNA viruses in community-acquired childhood pneumonia in semi-urban Nepal; a cross-sectional study

<p>Abstract</p> <p>Background</p> <p>Pneumonia is among the main causes of illness and death in children <5 years of age. There is a need to better describe the epidemiology of viral community-acquired pneumonia (CAP) in developing countries.</p> <p>Methods</p> <p>From July 2004 to June 2007, we examined nasopharyngeal aspirates (NPA) from 2,230 cases of pneumonia (World Health Organization criteria) in children 2 to 35 months old recruited in a randomized trial of zinc supplementation at a field clinic in Bhaktapur, Nepal. The specimens were examined for respiratory syncytial virus (RSV), influenza virus type A (InfA) and B (InfB), parainfluenza virus types 1, 2 and 3 (PIV1, PIV2, and PIV3), and human metapneumovirus (hMPV) using a multiplex reverse transcriptase polymerase chain reaction (PCR) assay.</p> <p>Results</p> <p>We identified 919 virus isolates in 887 (40.0%) of the 2,219 NPA specimens with a valid PCR result, of which 334 (15.1%) yielded RSV, 164 (7.4%) InfA, 129 (5.8%) PIV3, 98 (4.4%) PIV1, 93 (4.2%) hMPV, 84 (3.8%) InfB, and 17 (0.8%) PIV2. CAP occurred in an epidemic pattern with substantial temporal variation during the three years of study. The largest peaks of pneumonia occurrence coincided with peaks of RSV infection, which occurred in epidemics during the rainy season and in winter. The monthly number of RSV infections was positively correlated with relative humidity (<it>r</it>_{<it>s </it>}= 0.40, <it>P </it>= 0.01), but not with temperature or rainfall. An hMPV epidemic occurred during one of the three winter seasons and the monthly number of hMPV cases was also associated with relative humidity (<it>r</it>_{<it>s </it>}= 0.55, <it>P </it>= 0.0005).</p> <p>Conclusion</p> <p>Respiratory RNA viruses were detected from NPA in 40% of CAP cases in our study. The most commonly isolated viruses were RSV, InfA, and PIV3. RSV infections contributed substantially to the observed CAP epidemics. The occurrence of viral CAP in this community seemed to reflect more or less overlapping micro-epidemics with several respiratory viruses, highlighting the challenges of developing and implementing effective public health control measures.</p

The relationship of interacting immunological components in dengue pathogenesis

The World Health Organization (WHO) estimates that there are over 50 million cases of dengue fever reported annually and approximately 2.5 billion people are at risk. Mild dengue fever presents with headache, fever, rash, myalgia, osteogenic pain, and lethargy. Severe disease can manifest as dengue shock syndrome (DSS) or dengue hemorrhagic fever (DHF). Symptoms of DSS/DHF are leukopenia, low blood volume and pressure encephalitis, cold and sweaty skin, gastrointestinal bleeding, and spontaneous bleeding from gums and nose. Currently, there are no therapeutics available beyond supportive care and untreated complicated dengue fever can have a 50% mortality rate. According to WHO DSS/DHF is the leading cause of childhood mortality in some Asian countries. Dendritic cells are professional antigen presenting cells that are primary targets in a dengue infection. Dengue binds to Dendritic Cell-Specific Intercellular adhesion molecule-3-Grabbing Non-integrin (DC-SIGN). DC-SIGN has a high affinity for ICAM3 which is expressed in activating T-cells. Previous studies have demonstrated an altered T-cell phenotype expressed in dengue infected patients that could be potentially mediated by dengue-infected DCs

A novel, fast, HMM-with-Duration implementation – for application with a new, pattern recognition informed, nanopore detector

<p>Abstract</p> <p>Background</p> <p>Hidden Markov Models (HMMs) provide an excellent means for structure identification and feature extraction on stochastic sequential data. An HMM-with-Duration (HMMwD) is an HMM that can also exactly model the hidden-label length (recurrence) distributions – while the regular HMM will impose a best-fit geometric distribution in its modeling/representation.</p> <p>Results</p> <p>A Novel, Fast, HMM-with-Duration (HMMwD) Implementation is presented, and experimental results are shown that demonstrate its performance on two-state synthetic data designed to model Nanopore Detector Data. The HMMwD experimental results are compared to (i) the ideal model and to (ii) the conventional HMM. Its accuracy is clearly an improvement over the standard HMM, and matches that of the ideal solution in many cases where the standard HMM does not. Computationally, the new HMMwD has all the speed advantages of the conventional (simpler) HMM implementation. In preliminary work shown here, HMM feature extraction is then used to establish the first pattern recognition-informed (PRI) sampling control of a Nanopore Detector Device (on a "live" data-stream).</p> <p>Conclusion</p> <p>The improved accuracy of the new HMMwD implementation, at the same order of computational cost as the standard HMM, is an important augmentation for applications in gene structure identification and channel current analysis, especially PRI sampling control, for example, where speed is essential. The PRI experiment was designed to inherit the high accuracy of the well characterized and distinctive blockades of the DNA hairpin molecules used as controls (or blockade "test-probes"). For this test set, the accuracy inherited is 99.9%.</p

PhagoSight: an open-source MATLAB® package for the analysis of fluorescent neutrophil and macrophage migration in a zebrafish model

Neutrophil migration in zebrafish larvae is increasingly used as a model to study the response of these leukocytes to different determinants of the cellular inflammatory response. However, it remains challenging to extract comprehensive information describing the behaviour of neutrophils from the multi-dimensional data sets acquired with widefield or confocal microscopes. Here, we describe PhagoSight, an open-source software package for the segmentation, tracking and visualisation of migrating phagocytes in three dimensions. The algorithms in PhagoSight extract a large number of measurements that summarise the behaviour of neutrophils, but that could potentially be applied to any moving fluorescent cells. To derive a useful panel of variables quantifying aspects of neutrophil migratory behaviour, and to demonstrate the utility of PhagoSight, we evaluated changes in the volume of migrating neutrophils. Cell volume increased as neutrophils migrated towards the wound region of injured zebrafish. PhagoSight is openly available as MATLAB® m-files under the GNU General Public License. Synthetic data sets and a comprehensive user manual are available from http://www.phagosight.org