Cutting edge: murine cytomegalovirus induces a polyfunctional CD4 T cell response

CD4 T lymphocytes regulate the adaptive immune response to most viruses, both by providing help to CD8 T cells and B cells as well as through direct antiviral activity. Currently, no mouse cytomegalovirus (MCMV)-specific CD4 T cell responses are known. In this study, we identify and characterize 15 I-A(b)-restricted CD4 T cell responses specific for MCMV epitopes. CD4 T cells accumulate to high levels in the spleen and lungs during acute infection and produce multiple cytokines (IFN-gamma, TNF, IL-2, IL-10, and IL-17). Interestingly, IL-17 and IFN-gamma production within epitope-specific cells was found to be mutually exclusive. CD4 T cells recognizing a peptide derived from m09 were only detectable at later times of infection and displayed a unique cytokine production profile. In total, this study reveals that the MCMV-specific CD4 T cell response is complex and functionally diverse, highlighting its important role in controlling this persistent pathogen.     

Operant Learning of Drosophila at the Torque Meter

For experiments at the torque meter, flies are kept on standard fly medium at 25°C and 60% humidity with a 12hr light/12hr dark regime. A standardized breeding regime assures proper larval density and age-matched cohorts. Cold-anesthetized flies are glued with head and thorax to a triangle-shaped hook the day before the experiment. Attached to the torque meter via a clamp, the fly''s intended flight maneuvers are measured as the angular momentum around its vertical body axis. The fly is placed in the center of a cylindrical panorama to accomplish stationary flight. An analog to digital converter card feeds the yaw torque signal into a computer which stores the trace for later analysis. The computer also controls a variety of stimuli which can be brought under the fly''s control by closing the feedback loop between these stimuli and the yaw torque trace. Punishment is achieved by applying heat from an adjustable infrared laser.     

Prior residence, territory quality and life-history strategies in juvenile Atlantic salmon (Salmo salar L.)

Three groups of juvenile salmon were introduced sequentially into an artificial stream to investigate the effects of prior residence on behaviour and territory choice. Almost half of the first group obtained and defended distinct territories, the other half being constrained to an area approximately the size of one large territory. All of the fish in the subsequent groups, bar one, were also constrained to the same site. Since the fish were of similar size, prior residence alone seemed to influence which individuals obtained territories. However, within the first group, the fish that obtained territories were larger and more aggressive. The territorial fish did not appear to choose the most profitable territories, although they had the greatest opportunity to do so. Since juvenile salmon emerge from their gravel nests fairly synchronously, a time constraint on site sampling is hypothesised: there may be a risk in taking time to sample sites, since these same sites may become occupied with conspecifics. However, fish with territories fed at faster rates than non-territorial fish, possibly because of reduced competition for prey items. Consequently, fish from the first group (containing most of the territorial fish) grew faster than the other two groups. Moreover, most of the territory holders, but only one of the non-territorial fish, reached the threshold size that increases their probability of smolting the following year. This suggests that ability to obtain a defensible territory, primarily through prior residence, influences the age at which juvenile salmon can migrate to sea.     

A systematic assessment of MHC class II peptide binding predictions and evaluation of a consensus approach

The identification of MHC class II restricted peptide epitopes is an important goal in immunological research. A number of computational tools have been developed for this purpose, but there is a lack of large-scale systematic evaluation of their performance. Herein, we used a comprehensive dataset consisting of more than 10,000 previously unpublished MHC-peptide binding affinities, 29 peptide/MHC crystal structures, and 664 peptides experimentally tested for CD4+ T cell responses to systematically evaluate the performances of publicly available MHC class II binding prediction tools. While in selected instances the best tools were associated with AUC values up to 0.86, in general, class II predictions did not perform as well as historically noted for class I predictions. It appears that the ability of MHC class II molecules to bind variable length peptides, which requires the correct assignment of peptide binding cores, is a critical factor limiting the performance of existing prediction tools. To improve performance, we implemented a consensus prediction approach that combines methods with top performances. We show that this consensus approach achieved best overall performance. Finally, we make the large datasets used publicly available as a benchmark to facilitate further development of MHC class II binding peptide prediction methods.     

Double dissociation of PKC and AC manipulations on operant and classical learning in Drosophila

Learning about relationships between stimuli (i.e., classical conditioning [1]) and learning about consequences of one's own behavior (i.e., operant conditioning [2]) constitute the major part of our predictive understanding of the world. Since these forms of learning were recognized as two separate types 80 years ago [3], a recurrent concern has been the issue of whether one biological process can account for both of them [4, 5, 6, 7, 8, 9]. Today, we know the anatomical structures required for successful learning in several different paradigms, e.g., operant and classical processes can be localized to different brain regions in rodents [9] and an identified neuron in Aplysia shows opposite biophysical changes after operant and classical training, respectively [5]. We also know to some detail the molecular mechanisms underlying some forms of learning and memory consolidation. However, it is not known whether operant and classical learning can be distinguished at the molecular level. Therefore, we investigated whether genetic manipulations could differentiate between operant and classical learning in Drosophila. We found a double dissociation of protein kinase C and adenylyl cyclase on operant and classical learning. Moreover, the two learning systems interacted hierarchically such that classical predictors were learned preferentially over operant predictors.     

Implantation of Engineered Tissue in the Rat Heart

Rodent surgery is often an important component in assessing the utility of engineered tissues. A wide variety of surgical procedures can be performed in common laboratory rats or mice and these quite frequently serve as an intermediate step between bench-top experiments and large animal testing or human trials. Given that rodents provide an established, cost-effective, and physiologically-relevant model system in which to test novel combinations of scaffolding materials and cells, they are particularly well-suited for cardiovascular tissue engineering studies. Presently, we describe an open-heart surgical procedure to implant engineered tissue containing myogenic progenitor cells in the atrioventricular (AV) groove of a rat heart. These implants are intended to create an electrical conduit between the right atrium and right ventricle with the ultimate goal of providing an alternative treatment to conventional pacemaker implantation in pediatric patients with complete heart block[1]. The engineered tissue is implanted in the AV-groove by means of a thoracotomy. For our purposes, Lewis rats are anesthetized and invasively ventilated to maintain positive airway pressure during the sterile surgical procedure. The approach to the heart is performed by a right thoracotomy through an antero-lateral incision at the 5^th intercostal space. The tissue construct is fixed in the AV groove using a single 7-0 Prolene suture and positioned between the right ventricle and atrium at the ventral portion of the heart. The epicardium is partially removed to allow direct contact between the recipient myocardial cells and those contained in the engineered tissue. Following implantation, the chest wall is closed in layers, any pneumothorax is evacuated, and the animal is extubated and treated with analgesic.Download video file.^{(95M, mp4)}