CARACTERIZACIÓN DE NANOPARTÍCULAS DE ÓXIDO DE HIERRO

Due to the only limited basic science knowledge behind SRL-172 it is difficult to establish fruitful directions of research into this product. The approach adopted here involved the collation of the present knowledge base in both the immunology and clinical efficacies of this preparation of heat-killed M. vaccae and the development of hypotheses based upon these that could then be tested. Such hypotheses studied in this thesis were effects on less well understood lymphocyte subsets, especially yô TCR cells, NK cells and NKT cells, and the effects on the humoral responses to the heat shock protein hsp60/65. The latter sub-study also involved the amplification and sequencing of the hsp65 gene of the vaccine strain

of M. vaccae. Other approaches that were investigated, but have not been included

in this thesis, were potential effects on cross presentation of exogenous antigen onto MHC class I molecules by dendritic and other antigen-presenting cells, effects on double negative (DN) T cells (negative for CD4, CDS, yÔTCR and CD56 but positive for the T cell marker CD3), and the effects of in-vivo

vaccination in healthy volunteers on cytokine profiles of peripheral blood mononuclear cells (as assessed by RT-PCR).

The overall results of the work contained in this thesis can be summarised as the demonstration of alterations in proliferation and activation markers on yÔ T cells (in particular effects upon CD25, CD69, CD86 and cytokine production), and on CD56+ NK cells (principally effects upon proliferation and the activation markers CD25 and CD69, with trends to increased cytokine production) as a result of exposure to heat-killed M. vaccae. The relevance of these findings to the in-vivo

activities of SRL-172 is to be further investigated. Some effects of SRL-172 immunisation on the humoral responses to the 60kDa heat shock protein family were also demonstrated. Principally these were the relative increase in IgG2a titres to the human (but not the mycobacterial) hsp60/65 homologue in rats that received SRL-172 prior to hsp65 immunisation, and the apparent correlation between aortic contractility and IgG2a titres to hsp60 in those rodents that received both hsp65

and high-dose SRL-172 vaccination. The relevances of these results to the in vivo

efficacies of this vaccine have yet to be fully established.

There are several different ways that investigation into the basic immunological mechanisms of action of heat-killed M. vaccae may be taken forward. One such, that is presently being set up within the laboratory, is the investigation of differential gene induction by SRL-172. Differential display RT-PCR (DD-PCR) is a technique that permits the identification of such up- and down-regulated genes and, though it frequently produces false positive results, has the advantages of relative simplicity and the use of established techniques. Other methods, such as selective subtractive hybridisation, might also be used. These techniques would be expected to highlight certain genes, and thereby potentially certain cell types, that then merit further investigation. Also, as more stringent and larger clinical trials and basic immunological studies are completed, further information will be gained on efficacies and other parameters that might suggest further, different approaches.

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