CD8+ T-cell priming and boosting: more antigen-presenting DC, or more antigen per DC?

RNA transfection is a standard method to load dendritic cells (DC) with antigen for therapeutic cancer vaccination. While electroporation yields high transfection efficiency and satisfying expression levels, lipofection results in only few cells expressing high amounts of antigen. We compared antigen loading of human monocyte-derived DC by MelanA RNA electroporation and lipofection. No differences in phenotype or migrational capacity were detected, but lipofected DC induced stronger cytokine secretion by antigen-specific T cells and were superior in priming and boosting of MelanA-specific CD8⁺ T cells. Interestingly, T cells stimulated with the differently transfected DC did not differ in their functional avidity. To determine whether the amount of antigen per cell is indeed responsible for the superiority of the lipofected DC, we increased the amount of MelanA RNA fivefold and mixed those DC with mock-electroporated ones to mimic the antigen distribution of lipofected cells. This significantly improved the stimulatory capacity, indicating that indeed the amount of antigen per cell seems to be the responsible feature for the observed superiority of lipofected DCs. These data suggest that a few DC that express high amounts of antigen are more immunogenic than many DC expressing lower amounts, although this needs to be tested in a two-armed immunogenicity trial.     

Impacts of eriophyoid gall mites on arctic willow in a rapidly changing Arctic

Arctic plants and herbivores are subject to ongoing climatic changes that are more rapid and extreme than elsewhere on the planet, and thus it is pivotal to understand the arctic plant-herbivore interactions in a global change context. We examined how infestation by an eriophyoid gall mite affects the circumpolar shrub Salix arctica, and how the effects vary across vegetation types. Specifically, we compared multiple leaf characteristics (leaf area, biomass, nutrient levels, δ¹⁵N and δ¹³C, and stress and performance of the photosynthetic apparatus) of infested leaves to those of un-infested leaves. Furthermore, we examined how altered environmental conditions, here experimentally manipulated levels of temperature, water and nutrients, shading, and UV-B radiation, affect the prevalence, density, and intensity of gall mite infestation and its impacts on S. arctica. Infested leaves were smaller in area and biomass and had lower nitrogen and carbon pools. However, their carbon concentration was higher, possibly because the galls acted as carbon sinks. The smaller photosynthetic area and lower nutrient content caused increased stress on the photosynthetic apparatus in infested leaves. The remaining leaf tissue responded with a higher photosynthetic performance, although there were indications of a general reduction in photosynthesis. Female leaves were more affected than male leaves. The experimental manipulations of environmental conditions did not affect the gall prevalence, density, or intensity on S. arctica leaves. Rather, plants responded positively to the treatments, reducing the effects of the galls to in-significance. This suggests a higher tolerance and defense against gall mites under future climate conditions.     

Phylogeny of the Bacillariaceae with emphasis on the genus Pseudo-nitzschia (Bacillariophyceae) based on partial LSU rDNA

In order to elucidate the phylogeny and evolutionary history of the Bacillariaceae we conducted a phylogenetic analysis of 42 species (sequences were determined from more than two strains of many of the Pseudo-nitzschia species) based on the first 872 base pairs of nuclear-encoded large subunit (LSU) rDNA, which include some of the most variable domains. Four araphid genera were used as the outgroup in maximum likelihood, parsimony and distance analyses. The phylogenetic inferences revealed the Bacillariaceae as monophyletic (bootstrap support ≥90%). A clade comprising Pseudo-nitzschia, Fragilariopsis and Nitzschia americana (clade A) was supported by high bootstrap values (≥94%) and agreed with the morphological features revealed by electron microscopy. Data for 29 taxa indicate a subdivision of clade A, one clade comprising Pseudo-nitzschia species, a second clade consisting of Pseudo-nitzschia species and Nitzschia americana, and a third clade comprising Fragilariopsis species. Pseudo-nitzschia as presently defined is paraphyletic and emendation of the genus is probably needed. The analyses suggested that Nitzschia is not monophyletic, as expected from the great morphological diversity within the genus. A cluster characterized by possession of detailed ornamentation on the frustule is indicated. Eighteen taxa (16 within the Bacillariaceae) were tested for production of domoic acid, a neurotoxic amino acid. Only P. australis, P. multiseries and P. seriata produced domoic acid, and these clustered together in all analyses. Since Nitzschia navis-varingica also produces domoic acid, but is distantly related to the cluster comprising the Pseudo-nitzschia domoic acid producers, it is most parsimonious to suggest that the ability of species in the Bacillariaceae to produce domoic acid has evolved at least twice.     

Techniques and applications of NMR to membrane proteins (Review)

The fact that membrane proteins are notoriously difficult to analyse using standard protocols for atomic-resolution structure determination methods have motivated adaptation of these techniques to membrane protein studies as well as development of new technologies. With this motivation, liquid-state nuclear magnetic resonance (NMR) has recently been used with success for studies of peptides and membrane proteins in detergent micelles, and solid-state NMR has undergone a tremendous evolution towards characterization of membrane proteins in native membrane and oriented phospholipid bilayers. In this mini-review, we describe some of the technological challenges behind these efforts and provide examples on their use in membrane biology.     

Spectrophotometric measurements of human tissues for the detection of subjacent blood vessels in an endonasal endoscopic surgical approach

Thin slices of human tissues are characterized concerning reflection and transmission in a wavelength range from 400 to 1700 nm. The results are primarily useful to find a wavelength for the detection of subjacent blood vessels during surgical procedures, especially neurological surgery. The measurements have been conducted using a customized measuring station, utilizing two halogen bulb lamps and two spectrometers. This paper focuses on creating a data base with the optical properties of artery, brain, bone, nasal mucosa, and nerve. The spectral distributions are compared among each other, similarities and differences are pointed out. Each tissue has got unique spectral characteristics, whereas typical absorption bands can be found in the overall tissues, especially hemoglobin and water absorption bands. The reflectivity maxima are typically located in the red or near‐infrared. All the transmission maxima are located between 1075 nm and 1100 nm. The measurements have been conducted at the Institute of Anatomy at the University of Leipzig. (© 2013 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Environmental Factors Associated with Disease Progression after the First Demyelinating Event: Results from the Multi-Center SET Study

Objectives

To investigate the associations of environmental MS risk factors with clinical and MRI measures of progression in high-risk clinically isolated syndromes (CIS) after the first demyelinating event.

Methods

We analyzed 211 CIS patients (age: 28.9±7.8 years) enrolled in the SET study, a multi-center study of high-risk CIS patients. Pre-treatment samples were analyzed for IgG antibodies against cytomegalovirus (anti-CMV), Epstein Barr virus (EBV) early nuclear antigen-1 (EBNA-1), viral capsid antigen (VCA), early antigen-diffuse (EA-D), 25 hydroxy-vitamin D3 and cotinine levels and HLA DRB1*1501 status. The inclusion criteria required evaluation within 4 months of the initial demyelinating event, 2 or more brain MRI lesions and the presence of two or more oligoclonal bands in cerebrospinal fluid. All patients were treated with interferon-beta. Clinical and MRI assessments were obtained at baseline, 6, 12, and 24 months.

Results

The time to first relapse decreased and the number of relapses increased with anti-CMV IgG positivity. Smoking was associated with increased number and volume of contrast-enhancing lesions (CEL) during the 2-year period. The cumulative number of CEL and T2 lesions during the 2-year period was greater for individuals in the highest quartile of anti-EBV VCA IgG antibodies. The percent loss of brain volume was increased for those in the highest quartile of with anti-EBV VCA IgG antibodies.

Conclusions

Relapses in CIS patients were associated with CMV positivity whereas anti-EBV VCA positivity was associated with progression on MRI measures, including accumulation of CEL and T2 lesions and development of brain atrophy.     

Transgenic Fluorescent Plasmodium cynomolgi Liver Stages Enable Live Imaging and Purification of Malaria Hypnozoite-Forms

A major challenge for strategies to combat the human malaria parasite Plasmodium vivax is the presence of hypnozoites in the liver. These dormant forms can cause renewed clinical disease after reactivation through unknown mechanisms. The closely related non-human primate malaria P. cynomolgi is a frequently used model for studying hypnozoite-induced relapses. Here we report the generation of the first transgenic P. cynomolgi parasites that stably express fluorescent markers in liver stages by transfection with novel DNA-constructs containing a P. cynomolgi centromere. Analysis of fluorescent liver stages in culture identified, in addition to developing liver-schizonts, uninucleate persisting parasites that were atovaquone resistant but primaquine sensitive, features associated with hypnozoites. We demonstrate that these hypnozoite-forms could be isolated by fluorescence-activated cell sorting. The fluorescently-tagged parasites in combination with FACS-purification open new avenues for a wide range of studies for analysing hypnozoite biology and reactivation.