Blocking KCa3.1 Channels Increases Tumor Cell Killing by a Subpopulation of Human Natural Killer Lymphocytes

Natural killer (NK) cells are large granular lymphocytes that participate in both innate and adaptive immune responses against tumors and pathogens. They are also involved in other conditions, including organ rejection, graft-versus-host disease, recurrent spontaneous abortions, and autoimmune diseases such as multiple sclerosis. We demonstrate that human NK cells express the potassium channels Kv1.3 and KCa3.1. Expression of these channels does not vary with expression levels of maturation markers but varies between adherent and non-adherent NK cell subpopulations. Upon activation by mitogens or tumor cells, adherent NK (A-NK) cells preferentially up-regulate KCa3.1 and non-adherent (NA-NK) cells preferentially up-regulate Kv1.3. Consistent with this different phenotype, A-NK and NA-NK do not display the same sensitivity to the selective KCa3.1 blockers TRAM-34 and NS6180 and to the selective Kv1.3 blockers ShK-186 and PAP-1 in functional assays. Kv1.3 block inhibits the proliferation and degranulation of NA-NK cells with minimal effects on A-NK cells. In contrast, blocking KCa3.1 increases the degranulation and cytotoxicity of A-NK cells, but not of NA-NK cells. TRAM-34, however, does not affect their ability to form conjugates with target tumor cells, to migrate, or to express chemokine receptors. TRAM-34 and NS6180 also increase the proliferation of both A-NK and NA-NK cells. This results in a TRAM-34-induced increased ability of A-NK cells to reduce in vivo tumor growth. Taken together, our results suggest that targeting KCa3.1 on NK cells with selective blockers may be beneficial in cancer immunotherapy.     

Functional Characterisation of the Maturation of the Blood-Brain Barrier in Larval Zebrafish

Zebrafish are becoming increasingly popular as an organism in which to model human disease and to study the effects of small molecules on complex physiological and pathological processes. Since larvae are no more than a few millimetres in length, and can live in volumes as small as 100 microliters, they are particularly amenable to high-throughput and high content compound screening in 96 well plate format. There is a growing literature providing evidence that many compounds show similar pharmacological effects in zebrafish as they do in mammals, and in particular humans. However, a major question regarding their utility for small molecule screening for neurological conditions is whether a molecule will reach its target site within the central nervous system. Studies have shown that Claudin-5 and ZO-1, tight-junction proteins which are essential for blood-brain barrier (BBB) integrity in mammals, can be detected in some cerebral vessels in zebrafish from 3 days post-fertilisation (d.p.f.) onwards and this timing coincides with the retention of dyes, immunoreactive tracers and fluorescent markers within some but not all cerebral vessels. Whilst these findings demonstrate that features of a BBB are first present at 3 d.p.f., it is not clear how quickly the zebrafish BBB matures or how closely the barrier resembles that of mammals. Here, we have combined anatomical analysis by transmission electron microscopy, functional investigation using fluorescent markers and compound uptake using liquid chromatography/tandem mass spectrometry to demonstrate that maturation of the zebrafish BBB occurs between 3 d.p.f. and 10 d.p.f. and that this barrier shares both structural and functional similarities with that of mammals.     

Neutral Sphingomyelinase in Physiological and Measles Virus Induced T Cell Suppression

T cell paralysis is a main feature of measles virus (MV) induced immunosuppression. MV contact mediated activation of sphingomyelinases was found to contribute to MV interference with T cell actin reorganization. The role of these enzymes in MV-induced inhibition of T cell activation remained equally undefined as their general role in regulating immune synapse (IS) activity which relies on spatiotemporal membrane patterning. Our study for the first time reveals that transient activation of the neutral sphingomyelinase 2 (NSM2) occurs in physiological co-stimulation of primary T cells where ceramide accumulation is confined to the lamellum (where also NSM2 can be detected) and excluded from IS areas of high actin turnover. Genetic ablation of the enzyme is associated with T cell hyper-responsiveness as revealed by actin dynamics, tyrosine phosphorylation, Ca²⁺-mobilization and expansion indicating that NSM2 acts to suppress overshooting T cell responses. In line with its suppressive activity, exaggerated, prolonged NSM2 activation as occurring in co-stimulated T cells following MV exposure was associated with aberrant compartmentalization of ceramides, loss of spreading responses, interference with accumulation of tyrosine phosphorylated protein species and expansion. Altogether, this study for the first time reveals a role of NSM2 in physiological T cell stimulation which is dampening and can be abused by a virus, which promotes enhanced and prolonged NSM2 activation to cause pathological T cell suppression.     

Interspecific Aggression and Resource Monopolization of the Invasive Ant Anoplolepis gracilipes in Malaysian Borneo

Invasions by introduced ant species can be ecologically destructive and affect a wide range of taxa, particularly native ants. Invasive ant species often numerically dominate ant communities and outperform native ant species in effective resource acquisition. Here, we describe interactions between the invasive ant Anoplolepis gracilipes (Smith) and resident ant species in disturbed habitats in NE Borneo. We measured interference competition abilities of A. gracilipes by performing arena bioassays between two A. gracilipes colonies and seven local ant species, and measured its effective resource competition at baits within supercolonies and at supercolony boundaries. Furthermore, we compared ant species diversity and composition at baits among (A) core areas of A. gracilipes supercolonies, (B) supercolony boundaries and (C) outside supercolonies. Anoplolepis gracilipes was behaviorally dominant over most ant species except Oecophylla smaragdina. Within supercolonies, A. gracilipes discovered all food baits first, and monopolized the vast majority throughout the course of the experiment. At supercolony boundaries, A. gracilipes discovered baits later than resident ant species, but subsequently monopolized half of the baits. Furthermore, the activity and diversity of the ant community within A. gracilipes supercolonies was lower than at its boundaries and outside supercolonies, and the ant communities differed significantly between infested and noninfested areas. Our study supports the hypothesis that successful establishment of A. gracilipes in anthropogenically disturbed habitats may negatively affect resident ant communities through high levels of direct interspecific aggression and almost complete monopolization of resources within high‐density supercolonies.     

Deviance-Related Responses along the Auditory Hierarchy: Combined FFR,MLR and MMN Evidence

The mismatch negativity (MMN) provides a correlate of automatic auditory discrimination in human auditory cortex that is elicited in response to violation of any acoustic regularity. Recently, deviance-related responses were found at much earlier cortical processing stages as reflected by the middle latency response (MLR) of the auditory evoked potential, and even at the level of the auditory brainstem as reflected by the frequency following response (FFR). However, no study has reported deviance-related responses in the FFR, MLR and long latency response (LLR) concurrently in a single recording protocol. Amplitude-modulated (AM) sounds were presented to healthy human participants in a frequency oddball paradigm to investigate deviance-related responses along the auditory hierarchy in the ranges of FFR, MLR and LLR. AM frequency deviants modulated the FFR, the Na and Nb components of the MLR, and the LLR eliciting the MMN. These findings demonstrate that it is possible to elicit deviance-related responses at three different levels (FFR, MLR and LLR) in one single recording protocol, highlight the involvement of the whole auditory hierarchy in deviance detection and have implications for cognitive and clinical auditory neuroscience. Moreover, the present protocol provides a new research tool into clinical neuroscience so that the functional integrity of the auditory novelty system can now be tested as a whole in a range of clinical populations where the MMN was previously shown to be defective.     

A New Method for the Separation of Different Types of Nematocysts from Scyphozoa and Investigation of Proteinaceous Toxins Utilizing Laser Catapulting and Subsequent Mass Spectrometry

Jellyfish have an increasing impact on marine ecology. Cnidocysts bearing stinging cells afford, amongst others, prey capture and defence. Several different types of stinging capsules are found in one species and they are supposed to have specific functions, e.g. paralysing prey or adhering to it. Due to these assumed different roles of the capsules, it is suggested that toxins, which are contained in the capsules, differ in composition. Analysis of distinct types of nematocysts requires an appropriate method for the separation of the different types. Mixtures of types of nematocysts were obtained of two species of jellyfish, Aurelia aurita and Cyanea lamarckii, by maceration of the tissue. These mixtures were treated with a method called laser microdissection and pressure catapulting (LMPC). Optimized maceration methods, which were firstly introduced as a method for this purpose, in conjunction with optimized LMPC parameters lead to sufficient amounts of separated capsules of individual types for subsequent mass-spectrometric analyses. In case of A. aurita, the resulting mass spectra had some constituents in common, whereas in the overall pattern, the two distinct nematocyst types differed.