Alphavirus Replicon Particles Expressing TRP-2 Provide Potent Therapeutic Effect on Melanoma through Activation of Humoral and Cellular Immunity

Background

Malignant melanoma is the deadliest form of skin cancer and is refractory to conventional chemotherapy and radiotherapy. Therefore alternative approaches to treat this disease, such as immunotherapy, are needed. Melanoma vaccine design has mainly focused on targeting CD8⁺ T cells. Activation of effector CD8⁺ T cells has been achieved in patients, but provided limited clinical benefit, due to immune-escape mechanisms established by advanced tumors. We have previously shown that alphavirus-based virus-like replicon particles (VRP) simultaneously activate strong cellular and humoral immunity against the weakly immunogenic melanoma differentiation antigen (MDA) tyrosinase. Here we further investigate the antitumor effect and the immune mechanisms of VRP encoding different MDAs.

Methodology/Principal Findings

VRP encoding different MDAs were screened for their ability to prevent the growth of the B16 mouse transplantable melanoma. The immunologic mechanisms of efficacy were investigated for the most effective vaccine identified, focusing on CD8⁺ T cells and humoral responses. To this end, ex vivo immune assays and transgenic mice lacking specific immune effector functions were used. The studies identified a potent therapeutic VRP vaccine, encoding tyrosinase related protein 2 (TRP-2), which provided a durable anti-tumor effect. The efficacy of VRP-TRP2 relies on a novel immune mechanism of action requiring the activation of both IgG and CD8⁺ T cell effector responses, and depends on signaling through activating Fcγ receptors.

Conclusions/Significance

This study identifies a VRP-based vaccine able to elicit humoral immunity against TRP-2, which plays a role in melanoma immunotherapy and synergizes with tumor-specific CD8⁺ T cell responses. These findings will aid in the rational design of future immunotherapy clinical trials.     

Development and regeneration of the retinotectal map in goldfish: a computational study

We present a simple computational model to study the interplay of activity-dependent and intrinsic processes thought to be involved in the formation of topographic neural projections. Our model consists of two input layers which project to one target layer. The connections between layers are described by a set of synaptic weights. These weights develop according to three interacting developmental rules: (i) an intrinsic fibre-target interaction which generates chemospecific adhesion between afferent fibres and target cells; (ii) an intrinsic fibre-fibre interaction which generates mutual selective adhesion between the afferent fibres; and (iii) an activity-dependent fibre-fibre interaction which implements Hebbian learning. Additionally, constraints are imposed to keep synaptic weights finite. The model is applied to a set of eleven experiments on the regeneration of the retinotectal projection in goldfish. We find that the model is able to reproduce the outcome of an unprecedented range of experiments with the same set of model parameters, including details of the size of receptive and projective fields. We expect this mathematical framework to be a useful tool for the analysis of developmental processes in general. <br>     

Virtual reality training of lucid dreaming

Metacognitive reflections on one''s current state of mind are largely absent during dreaming. Lucid dreaming as the exception to this rule is a rare phenomenon; however, its occurrence can be facilitated through cognitive training. A central idea of respective training strategies is to regularly question one''s phenomenal experience: is the currently experienced world real, or just a dream? Here, we tested if such lucid dreaming training can be enhanced with dream-like virtual reality (VR): over the course of four weeks, volunteers underwent lucid dreaming training in VR scenarios comprising dream-like elements, classical lucid dreaming training or no training. We found that VR-assisted training led to significantly stronger increases in lucid dreaming compared to the no-training condition. Eye signal-verified lucid dreams during polysomnography supported behavioural results. We discuss the potential mechanisms underlying these findings, in particular the role of synthetic dream-like experiences, incorporation of VR content in dream imagery serving as memory cues, and extended dissociative effects of VR session on subsequent experiences that might amplify lucid dreaming training during wakefulness.This article is part of the theme issue ‘Offline perception: voluntary and spontaneous perceptual experiences without matching external stimulation''.     

Inhibition of α-Glycosidase by Lippia dulcis Trevir. (Verbenaceae) Preparations,Quantification of Verbascoside,and Study of Its Molecular Docking

This study aimed to quantify verbascoside (VEB), perform molecular docking studies of VEB with the α-glucosidase (GL) of Bacillus stearothermophilus, and evaluate the inhibition of the enzyme by L. dulcis preparations. The substrate concentration and presence of reduced glutathione were evaluated for their effect on the in vitro inhibition of the GL enzyme. Assays were also performed in the presence and absence of simulated gastric fluid. The antidiabetic fractions 2 and 3 were the most inhibited GL, but their activity were significantly decreased in the presence of gastric fluid. Chromatographic analyses confirmed the predominant presence of VEB in the samples. The samples had VEB concentrations between 49.9 and 243.5 mg/g. Simulation of the molecular docking of VEB were consistent with its GL-inhibitory activity. It can conclude that the crude ethanol extract and fractions show inhibitory activity against the GL enzyme.     

Cholecystokinin increases cytosolic calcium in a subpopulation of cultured vagal afferent neurons

Imaging fluorescent measurements with fura 2 were used to examine cytosolic calcium signals induced by sulfated CCK octapeptide (CCK-8) in dissociated vagal afferent neurons from adult rat nodose ganglia. We found that 40% (184/465) of the neurons responded to CCK-8 with a transient increase in cytosolic calcium. The threshold concentration of CCK-8 for inducing the response varied from 0.01 to 100 nM. In most neurons (13/16) the response was eliminated by removing extracellular calcium. Depleting intracellular calcium stores with thapsigargin slightly augmented the response. Most neurons were unresponsive to nonsulfated CCK-8. The response was eliminated by the CCK-A receptor antagonist lorglumide. Low concentrations of JMV-180 had no effect; however, high concentrations of JMV-180 reduced responses to CCK-8. These results demonstrate that CCK acts at the low-affinity site of the CCK-A receptor to trigger the entry of extracellular calcium into vagal afferent neurons. Increased cytosolic calcium may participate in acute activation of vagal afferent neurons, or it may initiate long-term changes, which modulate future neuronal responses to sensory stimuli.     

Comparative study on digestive lipase activities on the self emulsifying excipient Labrasol, medium chain glycerides and PEG esters

Labrasol is a lipid-based self-emulsifying excipient used in the preparation of lipophilic drugs intended for oral delivery. It is mainly composed of PEG esters and glycerides with medium acyl chains, which are potential substrates for digestive lipases. The hydrolysis of Labrasol by porcine pancreatic extracts, human pancreatic juice and several purified digestive lipases was investigated in the present study. Classical human pancreatic lipase (HPL) and porcine pancreatic lipase, which are the main lipases involved in the digestion of dietary triglycerides, showed very low levels of activity on the entire Labrasol excipient as well as on separated fractions of glycerides and PEG esters. On the other hand, gastric lipase, pancreatic lipase-related protein 2 (PLRP2) and carboxyl ester hydrolase (CEH) showed high specific activities on Labrasol. These lipases were found to hydrolyze the main components of Labrasol (PEG esters and monoglycerides) used as individual substrates, whereas these esters were found to be poor substrates for HPL. The lipolytic activity of pancreatic extracts and human pancreatic juice on Labrasol(R) is therefore mainly due to the combined action of CEH and PLRP2. These two pancreatic enzymes, together with gastric lipase, are probably the main enzymes involved in the in vivo lipolysis of Labrasol taken orally.     

Corpus Christi strain-induced protection to Trypanosoma cruzi infection in C3H(He) mice: effective dose, time, route, and number of vaccinations

The study of the parameters affecting Corpus Christi strain-induced protection in C3H(He) mice against Brazil strain T. cruzi infection is reported herein. A dose of 10(7) Corpus Christi epimastigotes was found to be the most effective dose for protection. Vaccination of mice 5 days to 11 wk prior to infection was determined to be the optimal time interval for protection. The subcutaneous route for vaccination and infection provides the most effective protection to experimental animals. Multiple inoculations with Corpus Christi, whether live or freeze thawed, increased the protective effect only slightly. The Corpus Christi strain of T. cruzi has proved to be quite suitable in providing protection to highly susceptible C3H(He) mice against an infection with the virulent Brazil strain of T. cruzi.     

Binding of phosphorylated peptides and inhibition of their interaction with disease‐relevant human proteins by synthetic metal‐chelate receptors

The modulation of biological signal transduction pathways by masking phosphorylated amino acid residues represents a viable route toward pharmacologic protein regulation. Binding of phosphorylated amino acid residues has been achieved with synthetic metal‐chelate receptors. The affinity and selectivity of such receptors can be enhanced if combined with a second binding site. We demonstrate this principle with a series of synthetic ditopic metal‐chelate receptors, which were synthesized and investigated for their binding affinity to phosphorylated short peptides under conditions of physiological pH. The compounds showing highest affinity were subsequently used to inhibit the interaction of the human STAT1 protein to a peptide derived from the interferon‐γ receptor, and between the checkpoint kinase Chk2 and its preferred binding motif. Two of the investigated ditopic synthetic receptors show a significant increase in inhibition activity. The results show that regulation of protein function by binding to phosphorylated amino acids is possible. The introduction of additional binding sites into the synthetic receptors increases their affinity, but the flexibility of the structures investigated so far prohibited stringent amino acid sequence selectivity in peptide binding. Copyright © 2009 John Wiley & Sons, Ltd.     

The NECAP PHear domain increases clathrin accessory protein binding potential

AP-2 is a key regulator of the endocytic protein machinery driving clathrin-coated vesicle (CCV) formation. One critical function, mediated primarily by the AP-2 alpha-ear, is the recruitment of accessory proteins. NECAPs are alpha-ear-binding proteins that enrich on CCVs. Here, we have solved the structure of the conserved N-terminal region of NECAP 1, revealing a unique module in the pleckstrin homology (PH) domain superfamily, which we named the PHear domain. The PHear domain binds accessory proteins bearing FxDxF motifs, which were previously thought to bind exclusively to the AP-2 alpha-ear. Structural analysis of the PHear domain reveals the molecular surface for FxDxF motif binding, which was confirmed by site-directed mutagenesis. The reciprocal analysis of the FxDxF motif in amphiphysin I identified distinct binding requirements for binding to the alpha-ear and PHear domain. We show that NECAP knockdown compromises transferrin uptake and establish a functional role for NECAPs in clathrin-mediated endocytosis. Our data uncover a striking convergence of two evolutionarily and structurally distinct modules to recognize a common peptide motif and promote efficient endocytosis.