The aims of this project were to investigate the effect of immune complexes on mononuclear phagocytes in the presence or absence of infection with the human immunodeficiency virus (HIV). Mono Mac 6 (MM6) cells and human monocyte-derived macrophages (MDM) were used to compare the effect of chemical stimulants, cytokines and immune complexes on surface antigen expression. The same experiments were performed using HIV-infected cells to determine the effect of HIV infection on these parameters. Some of the stimulants especially IL-6, IL-10, TNF-alpha and HIV increased CD80 expressions, the effect being greater in MDM than MM6 cells. This has implications for antigen presentation. Cytokines caused the differentiation of MM6 cells and significantly increased or decreased surface antigen expressions. The MM6 cells express surface antigens at lower levels then MDM this indicates that MM6 cells need to differentiate before expressing surface antigens. The high standard deviations obtained meant that no significant change in surface antigen expression was seen in all HIV non-infected and infected MDM incubated with various immune complexes and HIV-sera. Comparing whole blood incubated with KLH and rabbit anti-human IgG showed that both seemed to produce IL-10 and IL-6 early. Only rabbit complexes stimulated TNF-a release. MDM incubated with IL-6, IL-10, and TNF-alpha showed increased expression of CD16 and CD80 only at day 3. However, HIV proteins (CHO) incubated in whole blood caused a significantly release of IL-6 at 8 hours with no detection of IL-10 and TNF-alpha. MM6 infected with HIV-1 Ba-L for 5 days showed increased expression of CD16 and CD11c, but reduced expression of the other antigens examined. Significant levels of IL-10 were released at 8 and 12 hours with a slight increase in IL-6, and TNF-alpha. HIV protein-containing (CHO) immune complexes slightly increased IL-6 secretion at 8 hours at which point, IL-10 production was high. HIV- infected cells incubated with HlV-sera showed a lack of TNF-alpha release but IL-6 and IL-10 was detected at 12 and 8 hours respectively. The detection of high levels of IL-10 and the inhibition of TNF-alpha production may stimulate progression to full blown AIDS.

The quality of marine bathing water is currently assessed by monitoring the levels of faecal indicator bacteria. Among other drawbacks, results are retrospective using the traditional culture based methods. A rapid method is thus needed as an early warning to bathers for bacterial contamination in marine bathing waters. Total lipopolysaccharide (LPS) was chosen here as a potential general biomarker for bacterial contamination. Levels of total LPS, measured using a Kinetic QCL™ Limulus Amebocyte Lysate (LAL) assay, highly correlated with enumerated Escherichia coli and Bacteroides species. Levels of LPS in excess of 50 EU mL-1 were found to equate with water that was unsuitable for bathing under the current European Union regulations. Results showed that monitoring the levels of total LPS has a potential applicability as a rapid method for screening the quality of marine bathing water. More importantly, the LAL assay overcome the retrospective results when using culture based assessment since the LAL assay takes less than 30 minutes. Although false positive events were not detected, the occurrence of a false positive has been hypothesised, hence a more specific faecal biomarker was also investigated. LPS of five Bacteroides species (B. fragilis, B. caccae, B. ovatus, B. xylanisolvens and B. finegoldii) isolated from marine bathing waters samples were successfully profiled and showed high similarity between isolates in LPS gel electrophoresis banding pattern. Similar results were shown when investigating the endotoxic activity of Bacteroides species with the Kinetic QCL™ LAL assay. The potential biological relevance of Bacteroides LPS was also investigated in cell culture models indicating that Bacteroides showed similar induction of proinflammatory cytokines (TNF-α, IL-6 and IL-1α) and generally the biological activity was approximately 100 fold less than E. coli LPS. In addition, an ELISA assay was designed for the detection of Bacteroides LPS. Results showed that the Bacteroides LPS has a high potential to be used as a faecal biomarker, however, further work is required to develop a fully functional assay. The potential biological relevance of LPS present in contaminated bathing waters was also investigated in cell culture models. Results showed that there is a significant difference in the production of proinflammatory cytokines in comparison to “clean” bathing waters. Thus, results suggest that the European Directive regulations should be extended to cover the levels of total LPS in bathing waters to assure safety to the users of marine recreational water.