Intracellular Cytokine Staining and Flow Cytometry: Considerations for Application in Clinical Trials of Novel Tuberculosis Vaccines.

Intracellular cytokine staining combined with flow cytometry is one of a number of assays designed to assess T-cell immune responses. It has the specific advantage of enabling the simultaneous assessment of multiple phenotypic, differentiation and functional parameters pertaining to responding T-cells, most notably, the expression of multiple effector cytokines. These attributes make the technique particularly suitable for the assessment of T-cell immune responses induced by novel tuberculosis vaccines in clinical trials. However, depending upon the particular nature of a given vaccine and trial setting, there are approaches that may be taken at different stages of the assay that are more suitable than other alternatives. In this paper, the Tuberculosis Vaccine Initiative (TBVI) TB Biomarker Working group reports on efforts to assess the conditions that will determine when particular assay approaches should be employed. We have found that choices relating to the use of fresh whole blood or peripheral blood mononuclear cells (PBMC) and frozen PBMC; use of serum-containing or serum-free medium; length of stimulation period and use of co-stimulatory antibodies can all affect the sensitivity of intracellular cytokine assays. In the case of sample material, frozen PBMC, despite some loss of sensitivity, may be more advantageous for batch analysis. We also recommend that for multi-site studies, common antibody panels, gating strategies and analysis approaches should be employed for better comparability.

A nested location-routing heuristic using route length estimation

A nested heuristic approach that uses route length approximation is proposed to solve the location-routing problem. A new estimation formula for route length approximation is also developed. The heuristic is evaluated empirically against the sequential method and a recently developed nested method for location routing problems. This testing is carried out on a set of problems of 400 customers and around 15 to 25 depots with good results.

Keeping it together: a guide for support staff working with people whose behaviour is challenging

This guide has been written for support staff, working with people with learning disabilities, who are new to challenging behaviour or facing it for the first time. It is a self-help guide and provides practical and realistic information on how direct care staff can develop strageties to support people whose behaviour is challenging and develop their skills in this area.

Evaluating the process of borosilicate glass capillaries used for fabrication of in-vitro fertilization (IVF) micro-pipettes

In this paper we investigate a number of gas flames for fire polishing borosilicate glass capillaries used in the manufacturing of IVF micro-pipettes. Hydrofluoric acid (HF) was also used as an alternative to finish the pipette end. Glass micro tools in the IVF industry are drawn from hollow glass capillaries of diameter 1 mm. These capillaries are cut manually to a length of 100 mm from hollow glass rods resulting in sharp and chipped edges. These capillaries are held in a customised holder having padding of soft silicone or rubber. Sharp and uneven edges of these capillaries pick up particles of rubber or soft silicone shavings, rendering them ineffective for IVF treatments. The working range of borosilicate glass is 800-1,200 degrees C. The experiments involved analysis of fire polishing process for borosilicate glass capillaries using candle, butane, propane, 2350 butane propane, oxyacetylene gas flames, finding the optimum distance of the capillary relative to the flame, optimum time for which the capillary should be held in the flame and optimum region of the flame which gives the required temperature range. The results show that 2350 butane propane gas mix is optimum for fire polishing of borosilicate glass capillaries. The paper is concluded by comparing the results of fire polishing with the results of acid polishing, in which HF of 1.6% concentration is used to etch the ends of the borosilicate glass pipettes.

Biological controls on resuspension and the relationship between particle size and the Kolmogorov length scale in a shallow coastal sea

Measurements of suspended particle matter (SPM) and turbulence have been obtained over five tidal surveys during spring and summer 2010 at station L4 (5025 degrees N 04.22 degrees W, depth 50 m), in the Western English Channel. The relationship between turbulence intensity and bed stress is explored, with an in-line holographic imaging system evaluating the extent to which material is resuspended. Image analysis allows for the identification of SPM above a size threshold of 200 pm, capturing particle variability across tidal cycles and the two seasons. Dissipation of turbulent kinetic energy, which exceeds 10(-5) W kg(-1), yields maximum values of bed stress of between 0.17 and 0.20 N m(-2), frequently resulting in the resuspension of material from the bed. Resuspension is shown to promote aggregation of SPM into flocs, where the size of such particles is theoretically determined by the Kolmogorov microscale, l(k). During the spring surveys, flocs of a size larger than lk were observed, though this was not repeated during summer. It is proposed that the presence of gelatinous, biological material in spring allows flocculated particles to exceed l(k). This suggests that under specific circumstances, the limiting factor on the growth of flocculated SPM is not only turbulence, as previously thought, but the presence or absence of certain types of biological particle.

Calorific Ash Carbon And Nitrogen-Content In Relation To Length And Dry-Weight Of Parathemisto-Gaudichaudi (Amphipoda Hyperiidea) In The North-East Atlantic Ocean

The calorific, ash, carbon and nitrogen content, length and dry weight were determined for the hyperiid Parathemisto gaudichaudi (Guerin). Regression equations for all these variables were determined so that they can be estimated by calculation from measurements of length of the hyperiid. Mean values for total nitrogen and carbon were 7.79±0.85% and 36.80±4.18% of the dry weight, respectively. The carbon to calorific equivalent for P. gaudichaudi was 10.37 kcal g-1 carbon (9.13 kcal g-1 when corrected for nitrogen). The calorific value for ash-free adult P. gaudichaudi was 5.138 kcal g-1±1.309 (4.510 kcal g-1 when corrected for nitrogen). This large variation in the calorific content (coefficient of variation of 25.84%) can be accounted for largely by variation in the ash content (coefficient of variation of 21.84%). The calorific value determined for P. gaudichaudi is similar to that measured for other carnivorous crustaceans and adds support to the hypothesis that animals with high calorific content have a low fecundity and an energy-rich store which can be used as a buffer during unfavourable periods in their life.