Safety and immunogenicity of the group B streptococcus vaccine AlpN in a placebo-controlled double-blind phase 1 trial.

Group B streptococcus (GBS) is a leading cause of life-threatening neonatal infections and subsets of adverse pregnancy outcomes. Essentially all GBS strains possess one allele of the alpha-like protein (Alp) family. A maternal GBS vaccine, consisting of the fused N-terminal domains of the Alps αC and Rib (GBS-NN), was recently demonstrated to be safe and immunogenic in healthy adult women. To enhance antibody responses to all clinically relevant Alps, a second-generation vaccine has been developed (AlpN), also containing the N-terminal domain of Alp1 and the one shared by Alp2 and Alp3. In this study, the safety and immunogenicity of AlpN is assessed in a randomized, double-blind, placebo-controlled, and parallel-group phase I study, involving 60 healthy non-pregnant women. AlpN is well tolerated and elicits similarly robust and persistent antibody responses against all four Alp-N-terminal domains, resulting in enhanced opsonophagocytic killing of all Alp serotypes covered by the vaccine.

Flow Cytometry Assay for Recycling of LFA-1 in T-lymphocytes.

To enable cells to move forward, cell surface integrins are internalized into an endosomal compartment and subsequently intracellularly transported to be re-exposed at a new site on the cell membrane. Leukocytes are the fastest migrating cell type in the human body, which express the leukocyte-specific integrin LFA-1. Here, we describe a flow cytometry-based assay that allows the quantification of LFA-1 internalization and its re-expression on the cell surface in T lymphocytes. An advantage of using flow cytometry-based assay over biochemical methods is the low number of needed cells. This protocol can be also used to measure recycling of other receptors.

Integrin Dependent RhoB Activation Assay Using Leukocytes.

To be able to migrate, leukocyte needs to re-use its adhesion molecules to move forward. These adhesion molecules are called integrins and are intracellularly transported via endocytosis and exocytosis in order to translocate to a new site on the cell membrane. The intracellular transportation is regulated by different small GTPases including RhoB. Here we describe an activation assay of RhoB in leukocytes migrating on ICAM-1Fc coated dishes using commercially available Rhoteikin coated agarose beads. Although this is a specific protocol for LFA-1 induced RhoB activation, it can also be used for RhoB activation induced by other soluble and insoluble factors.

RhoB controls the Rab11-mediated recycling and surface reappearance of LFA-1 in migrating T lymphocytes.

The regulation of cell adhesion and motility is complex and requires the intracellular trafficking of integrins to and from sites of cell adhesion, especially in fast-moving cells such as leukocytes. The Rab family of guanosine triphosphatases (GTPases) is essential for vesicle transport, and vesicles mediate intracellular integrin trafficking. We showed that RhoB regulates the vesicular transport of the integrin LFA-1 along the microtubule network in migrating T lymphocytes. Impairment in RhoB function resulted in the accumulation of both LFA-1 and the recycling endosomal marker Rab11 at the rear of migrating T lymphocytes and decreased the association between these molecules. T lymphocytes lacking functional RhoB exhibited impaired recycling and subsequently decreased surface amounts of LFA-1, leading to reduced T cell adhesion and migration mediated by the cell adhesion molecule ICAM-1 (intercellular adhesion molecule-1). We propose that vesicle-associated RhoB is a regulator of the Rab11-mediated recycling of LFA-1 to the cell surface, an event that is necessary for T lymphocyte motility.

EEG and MEG brain-computer interface for tetraplegic patients.

We characterized features of magnetoencephalographic (MEG) and electroencephalographic (EEG) signals generated in the sensorimotor cortex of three tetraplegics attempting index finger movements. Single MEG and EEG trials were classified offline into two classes using two different classifiers, a batch trained classifier and a dynamic classifier. Classification accuracies obtained with dynamic classifier were better, at 75%, 89%, and 91% in different subjects, when features were in the 0.5-3.0-Hz frequency band. Classification accuracies of EEG and MEG did not differ.

Beta cell specific probing with fluorescent exendin-4 is progressively reduced in type 2 diabetic mouse models.

Probes based on GLP-1R agonist exendin-4 have shown promise as in vivo ß cell tracers. However, questions remain regarding the ß cell specificity of exendin-4 probes, and it is unclear if the expression levels of the GLP-1R are affected in a type 2 diabetic state. Using in vivo probing followed by ex vivo imaging we found fluorescent exendin-4 probes to distinctly label the pancreatic islets in mice in a Glp-1r dependent manner. Furthermore, a co-localization study revealed a near 100 percent ß cell specificity with less than one percent probing in other analyzed cell types. We then tested if probing was affected in models of type 2 diabetes using the Lepr(db/db) (db/db) and the Diet-Induced Obese (DIO) mouse. Although nearly all ß cells continued to be probed, we observed a progressive decline in probing intensity in both models with the most dramatic reduction seen in db/db mice. This was paralleled by a progressive decrease in Glp-1r protein expression levels. These data confirm ß cell specificity for exendin-4 based probes in mice. Furthermore, they also suggest that GLP-1R targeting probes may provide a tool to monitor ß cell function rather than mass in type 2 diabetic mouse models.

Real-time trafficking and signaling of the glucagon-like peptide-1 receptor.

The glucagon-like peptide-1 incretin receptor (GLP-1R) of family B G protein-coupled receptors (GPCRs) is a major drug target in type-2-diabetes due to its regulatory effect on post-prandial blood-glucose levels. The mechanism(s) controlling GLP-1R mediated signaling are far from fully understood. A fundamental mechanism controlling the signaling capacity of GPCRs is the post-endocytic trafficking of receptors between recycling and degradative fates. Here, we combined microscopy with novel real-time assays to monitor both receptor trafficking and signaling in living cells. We find that the human GLP-1R internalizes rapidly and with similar kinetics in response to equipotent concentrations of GLP-1 and the stable GLP-1 analogues exendin-4 and liraglutide. Receptor internalization was confirmed in mouse pancreatic islets. GLP-1R is shown to be a recycling receptor with faster recycling rates mediated by GLP-1 as compared to exendin-4 and liraglutide. Furthermore, a prolonged cycling of ligand-activated GLP-1Rs was observed and is suggested to be correlated with a prolonged cAMP signal.

EEG-based brain-computer interface for tetraplegics.

Movement-disabled persons typically require a long practice time to learn how to use a brain-computer interface (BCI). Our aim was to develop a BCI which tetraplegic subjects could control only in 30 minutes. Six such subjects (level of injury C4-C5) operated a 6-channel EEG BCI. The task was to move a circle from the centre of the computer screen to its right or left side by attempting visually triggered right- or left-hand movements. During the training periods, the classifier was adapted to the user's EEG activity after each movement attempt in a supervised manner. Feedback of the performance was given immediately after starting the BCI use. Within the time limit, three subjects learned to control the BCI. We believe that fast initial learning is an important factor that increases motivation and willingness to use BCIs. We have previously tested a similar single-trial classification approach in healthy subjects. Our new results show that methods developed and tested with healthy subjects do not necessarily work as well as with motor-disabled patients. Therefore, it is important to use motor-disabled persons as subjects in BCI development.