Blockade of PD-1/PD-L1 interactions is proving an exciting, durable therapeutic modality in a range of cancers whereby T cells are released from checkpoint inhibition to revive their inherent ...anti-tumour activity. Here we have studied various ways to model ex vivo T cell function in order to compare the impact of the clinically utilised anti-PD-1 antibody, pembrolizumab (Keytruda) on the activation of human T cells: focussing on the release of pro-inflammatory IFNγ and anti-inflammatory IL-10 to assess functionality. Firstly, we investigated the actions of pembrolizumab in an acute model of T-cell activation with either immature or mature allogeneic dendritic cells (DCs); pembrolizumab enhanced IFNγ and IL-10 release from purified CD4+ T-cells in the majority of donors with a bias towards pro-inflammatory cytokine release. Next, we modelled the impact of pembrolizumab in settings of more chronic T-cell activation. In a 7-day antigen-specific response to EBV peptides, the presence of pembrolizumab resulted in a relatively modest increase in both IFNγ and IL-10 release. Where pembrolizumab was assessed against long-term stimulated CD4+ cells that had up-regulated the exhaustion markers TIM-3 and PD-1, there was a highly effective enhancement of the otherwise exhausted response to allogeneic DCs with respect to IFNγ production. By contrast, the restoration of IL-10 production was considerably more limited. Finally, to assess a direct clinical relevance we investigated the consequence of PD-1/PD-L1 blockade in the disease setting of dissociated cells from lung and colon carcinomas responding to allogeneic DCs: here, pembrolizumab once more enhanced IFNγ production from the majority of tumour preparations whereas, again, the increase in IL-10 release was modest at best. In conclusion, we have shown that the contribution of PD-1-revealed by using a canonical blocking antibody to interrupt its interaction with PD-L1-to the production of an exemplar pro- and anti-inflammatory cytokine, respectively, depends in magnitude and ratio on the particular stimulation setting and activation status of the target T cell. We have identified a number of in vitro assays with response profiles that mimic features of dissociated cell populations from primary tumours thereby indicating these represent disease-relevant functional assays for the screening of immune checkpoint inhibitors in current and future development. Such in vitro assays may also support patient stratification of those likely to respond to immuno-oncology therapies in the wider population.
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► Forty-one described opsin gene duplication events in ray-finned fish. ► Large opsin repertoires are products of recent and ancient gene duplication. ► Tandem duplication is more ...common than other modes of duplication. ► Gene duplicates are retained most often in the RH2 and LWS opsin subfamilies.
Opsin gene sequences were first reported in the 1980s. The goal of that research was to test the hypothesis that human opsins were members of a single gene family and that variation in human color vision was mediated by mutations in these genes. While the new data supported both hypotheses, the greatest contribution of this work was, arguably, that it provided the data necessary for PCR-based surveys in a diversity of other species. Such studies, and recent whole genome sequencing projects, have uncovered exceptionally large opsin gene repertoires in ray-finned fishes (taxon, Actinopterygii). Guppies and zebrafish, for example, have 10 visual opsin genes each. Here we review the duplication and divergence events that have generated these gene collections. Phylogenetic analyses revealed that large opsin gene repertories in fish have been generated by gene duplication and divergence events that span the age of the ray-finned fishes. Data from whole genome sequencing projects and from large-insert clones show that tandem duplication is the primary mode of opsin gene family expansion in fishes. In some instances gene conversion between tandem duplicates has obscured evolutionary relationships among genes and generated unique key-site haplotypes. We mapped amino acid substitutions at so-called key-sites onto phylogenies and this exposed many examples of convergence. We found that dN/dS values were higher on the branches of our trees that followed gene duplication than on branches that followed speciation events, suggesting that duplication relaxes constraints on opsin sequence evolution. Though the focus of the review is opsin sequence evolution, we also note that there are few clear connections between opsin gene repertoires and variation in spectral environment, morphological traits, or life history traits.
Abstract Traditional forensic DNA interpretation methods are restricted as they are unable to deal completely with complex low level or mixed DNA profiles. This type of data has become more prevalent ...as DNA typing technologies become more sensitive. In addition they do not make full use of the information available in peak heights. Existing methods of interpretation are often described as binary which describes the fact that the probability of the evidence is assigned as 0 or 1 (hence binary) (see for example 1 at 7.3.3). These methods are being replaced by more advanced interpretation methods such as continuous models. In this paper we describe a series of models that can be used to calculate expected values for allele and stutter peak heights, and their ratio SR . This model could inform methods which implement a continuous method for the interpretation of DNA profiling data.
Variable expression of visual pigment proteins (opsins) in cone photoreceptors of the vertebrate retina is a primary determinant of vision plasticity. Switches in opsin expression or variable ...co-expression of opsins within differentiated cones have been documented for a few rodents and fishes, but the extent of photoreceptor types affected and potential functional significance are largely unknown. Here, we show that both single and double cones in the retina of a flatfish, the starry flounder (Platichthys stellatus), undergo visual pigment changes through opsin switches or variable opsin co-expression. As the post-metamorphic juvenile (i.e., the young asymmetric flatfish with both eyes on one side of the body) grows from ~5 g to ~196 g, some single cones and one member of unequal double cones switched from a visual pigment with maximum wavelength of absorbance, λ
, at shorter wavelengths (437 nm and 527 nm) to one with longer λ
(456 nm and 545 nm, respectively) whereas other cones had intermediate visual pigments (λ
at 445 nm or 536 nm) suggesting co-expression of two opsins. The shift toward longer wavelength absorbing visual pigments was in line with maximizing sensitivity to the restricted light spectrum at greater depths and achromatic detection of overhead targets.
Previous studies investigating associations between white matter alterations and duration of temporal lobe epilepsy (TLE) have shown differing results, and were typically limited to univariate ...analyses of tracts in isolation. In this study, we apply a multivariate measure (the Mahalanobis distance), which captures the distinct ways white matter may differ in individual patients, and relate this to epilepsy duration. Diffusion MRI, from a cohort of 94 subjects (28 healthy controls, 33 left‐TLE and 33 right‐TLE), was used to assess the association between tract fractional anisotropy (FA) and epilepsy duration. Using ten white matter tracts, we analysed associations using the traditional univariate analysis (z‐scores) and a complementary multivariate approach (Mahalanobis distance), incorporating multiple white matter tracts into a single unified analysis. For patients with right‐TLE, FA was not significantly associated with epilepsy duration for any tract studied in isolation. For patients with left‐TLE, the FA of two limbic tracts (ipsilateral fornix, contralateral cingulum gyrus) were significantly negatively associated with epilepsy duration (Bonferonni corrected p < .05). Using a multivariate approach we found significant ipsilateral positive associations with duration in both left, and right‐TLE cohorts (left‐TLE: Spearman's ρ = 0.487, right‐TLE: Spearman's ρ = 0.422). Extrapolating our multivariate results to duration equals zero (i.e., at onset) we found no significant difference between patients and controls. Associations using the multivariate approach were more robust than univariate methods. The multivariate Mahalanobis distance measure provides non‐overlapping and more robust results than traditional univariate analyses. Future studies should consider adopting both frameworks into their analysis in order to ascertain a more complete understanding of epilepsy progression, regardless of laterality.
Previous studies investigating associations between white matter alterations and duration of temporal lobe epilepsy (TLE) have shown differing results, and were typically limited to univariate analyses of tracts in isolation. In this study, we apply a multivariate measure to capture the distinct ways white matter may differ in individual patients, and relate this to epilepsy duration.
Objective
Predicting postoperative seizure freedom using functional correlation networks derived from interictal intracranial electroencephalography (EEG) has shown some success. However, there are ...important challenges to consider: (1) electrodes physically closer to each other naturally tend to be more correlated, causing a spatial bias; (2) implantation location and number of electrodes differ between patients, making cross‐subject comparisons difficult; and (3) functional correlation networks can vary over time but are currently assumed to be static.
Methods
In this study, we address these three challenges using intracranial EEG data from 55 patients with intractable focal epilepsy. Patients additionally underwent preoperative magnetic resonance imaging (MRI), intraoperative computed tomography, and postoperative MRI, allowing accurate localization of electrodes and delineation of the removed tissue.
Results
We show that normalizing for spatial proximity between nearby electrodes improves prediction of postsurgery seizure outcomes. Moreover, patients with more extensive electrode coverage were more likely to have their outcome predicted correctly (area under the receiver operating characteristic curve > 0.9, P « 0.05) but not necessarily more likely to have a better outcome. Finally, our predictions are robust regardless of the time segment analyzed.
Significance
Future studies should account for the spatial proximity of electrodes in functional network construction to improve prediction of postsurgical seizure outcomes. Greater coverage of both removed and spared tissue allows for predictions with higher accuracy.
Median survival for glioblastoma (GBM) remains <15 months. Human cytomegalovirus (CMV) antigens have been identified in GBM but not normal brain, providing an unparalleled opportunity to subvert CMV ...antigens as tumor-specific immunotherapy targets. A recent trial in recurrent GBM patients demonstrated the potential clinical benefit of adoptive T-cell therapy (ATCT) of CMV phosphoprotein 65 (pp65)-specific T cells. However,
analyses from this study found no change in the capacity of CMV pp65-specific T cells to gain multiple effector functions or polyfunctionality, which has been associated with superior antitumor efficacy. Previous studies have shown that dendritic cells (DC) could further enhance tumor-specific CD8
T-cell polyfunctionality
when administered as a vaccine. Therefore, we hypothesized that vaccination with CMV pp65 RNA-loaded DCs would enhance the frequency of polyfunctional CMV pp65-specific CD8
T cells after ATCT. Here, we report prospective results of a pilot trial in which 22 patients with newly diagnosed GBM were initially enrolled, of which 17 patients were randomized to receive CMV pp65-specific T cells with CMV-DC vaccination (CMV-ATCT-DC) or saline (CMV-ATCT-saline). Patients who received CMV-ATCT-DC vaccination experienced a significant increase in the overall frequencies of IFNγ
, TNFα
, and CCL3
polyfunctional, CMV-specific CD8
T cells. These increases in polyfunctional CMV-specific CD8
T cells correlated (
= 0.7371,
= 0.0369) with overall survival, although we cannot conclude this was causally related. Our data implicate polyfunctional T-cell responses as a potential biomarker for effective antitumor immunotherapy and support a formal assessment of this combination approach in a larger randomized study.
A randomized pilot trial in patients with GBM implicates polyfunctional T-cell responses as a biomarker for effective antitumor immunotherapy.
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First published in 1999. Routledge is an imprint of Taylor & Francis, an informa company. The aim of this book is to approach Latino fiction from a wider perspective, and to cross the standard ...critical boundaries between Latino groups in order to focus upon the literary language of a collection of complicated novels and stories.
The epilepsies are commonly accompanied by widespread abnormalities in cerebral white matter. ENIGMA-Epilepsy is a large quantitative brain imaging consortium, aggregating data to investigate ...patterns of neuroimaging abnormalities in common epilepsy syndromes, including temporal lobe epilepsy, extratemporal epilepsy, and genetic generalized epilepsy. Our goal was to rank the most robust white matter microstructural differences across and within syndromes in a multicentre sample of adult epilepsy patients. Diffusion-weighted MRI data were analysed from 1069 healthy controls and 1249 patients: temporal lobe epilepsy with hippocampal sclerosis (n = 599), temporal lobe epilepsy with normal MRI (n = 275), genetic generalized epilepsy (n = 182) and non-lesional extratemporal epilepsy (n = 193). A harmonized protocol using tract-based spatial statistics was used to derive skeletonized maps of fractional anisotropy and mean diffusivity for each participant, and fibre tracts were segmented using a diffusion MRI atlas. Data were harmonized to correct for scanner-specific variations in diffusion measures using a batch-effect correction tool (ComBat). Analyses of covariance, adjusting for age and sex, examined differences between each epilepsy syndrome and controls for each white matter tract (Bonferroni corrected at P < 0.001). Across 'all epilepsies' lower fractional anisotropy was observed in most fibre tracts with small to medium effect sizes, especially in the corpus callosum, cingulum and external capsule. There were also less robust increases in mean diffusivity. Syndrome-specific fractional anisotropy and mean diffusivity differences were most pronounced in patients with hippocampal sclerosis in the ipsilateral parahippocampal cingulum and external capsule, with smaller effects across most other tracts. Individuals with temporal lobe epilepsy and normal MRI showed a similar pattern of greater ipsilateral than contralateral abnormalities, but less marked than those in patients with hippocampal sclerosis. Patients with generalized and extratemporal epilepsies had pronounced reductions in fractional anisotropy in the corpus callosum, corona radiata and external capsule, and increased mean diffusivity of the anterior corona radiata. Earlier age of seizure onset and longer disease duration were associated with a greater extent of diffusion abnormalities in patients with hippocampal sclerosis. We demonstrate microstructural abnormalities across major association, commissural, and projection fibres in a large multicentre study of epilepsy. Overall, patients with epilepsy showed white matter abnormalities in the corpus callosum, cingulum and external capsule, with differing severity across epilepsy syndromes. These data further define the spectrum of white matter abnormalities in common epilepsy syndromes, yielding more detailed insights into pathological substrates that may explain cognitive and psychiatric co-morbidities and be used to guide biomarker studies of treatment outcomes and/or genetic research.
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