Identification of major factors influencing ELISpot-based monitoring of cellular responses to antigens from Mycobacterium tuberculosis.

A number of different interferon-gamma ELISpot protocols are in use in laboratories studying antigen-specific immune responses. It is therefore unclear how results from different assays compare, and what factors most significantly influence assay outcome. One such difference is that some laboratories use a short in vitro stimulation period of cells before they are transferred to the ELISpot plate; this is commonly done in the case of frozen cells, in order to enhance assay sensitivity. Other differences that may be significant include antibody coating of plates, the use of media with or without serum, the serum source and the number of cells added to the wells. The aim of this paper was to identify which components of the different ELISpot protocols influenced assay sensitivity and inter-laboratory variation. Four laboratories provided protocols for quantifying numbers of interferon-gamma spot forming cells in human peripheral blood mononuclear cells stimulated with Mycobacterium tuberculosis derived antigens. The differences in the protocols were compared directly. We found that several sources of variation in assay protocols can be eliminated, for example by avoiding serum supplementation and using AIM-V serum free medium. In addition, the number of cells added to ELISpot wells should also be standardised. Importantly, delays in peripheral blood mononuclear cell processing before stimulation had a marked effect on the number of detectable spot forming cells; processing delay thus should be minimised as well as standardised. Finally, a pre-stimulation culture period improved the sensitivity of the assay, however this effect may be both antigen and donor dependent. In conclusion, small differences in ELISpot protocols in routine use can affect the results obtained and care should be given to conditions selected for use in a given study. A pre-stimulation step may improve the sensitivity of the assay, particularly when cells have been previously frozen.

No evidence of host specialization in a parasitic pea-crab exploiting two echinoid hosts

The pinnotherid crab Dissodactylus primitivus lives parasitically on 2 burrowingechinoid species, Meoma ventricosa and Plagiobrissus grandis. The fecundity of female crabsvaries between hosts, and is higher when parasitizing P. grandis than M. ventricosa. Moreover, thehosts present great variations in morphology (size and density of spines). These characteristicssuggest the potential to differentiate crabs according to host species. We investigated the genetic(microsatellites) and morphometric (outline analysis) differentiation of this parasitic crab between2 host species at 1 Jamaican site (Western Lagoon, Discovery Bay), and compared it with geographicdifferentiation among 4 sites along the north coast of Jamaica. Greater genetic differencesbetween parasites of the 2 sympatric hosts than between parasites of a single host at different geographiclocations would indicate host differentiation. Genetic analyses (microsatellites) did notdetect spatial differentiation (probably due to local hydrography) or differentiation according tohost species. This lack of host differentiation could be explained by mobility of adult crabsbetween hosts. However, there was weak but significant morphological differentiation betweenfemale crabs from the 2 hosts. This morphological difference may reflect constraints due to hostmorphology.

Monitoring SO2 emission at the Soufriere Hills Volcano: implications for changes in eruptive conditions

info:eu-repo/semantics/published