## 2 Results and Discussion
The analysis of all the tested samples (extracts and SPE-fractions) gave 40% of positive hits with the interesting result that 50% of the activity was detectable only after extract fractionation (Figure 2). In fact, SPE fractions contained less salt (mainly NaCl) than primary extracts, as measured according to the method proposed by [PERSON] and [PERSON] [20]. Consequently, the relative concentrations of the active products were enhanced in these samples, thus increasing the chances of positive response in the biological screening. This was marked with extracts of sponges that generally contained higher level of NaCl and probably other interfering components than those of protists. Furthermore, except for two samples (see below), we observed a general increase in activity of the fractions in comparison to extracts. \begin{table}
\begin{tabular}{c c c c c c} \hline \hline \multicolumn{2}{c}{**(1) Sample Preparation**} & \multicolumn{2}{c}{**(2) Column Equilibration**} & \multicolumn{2}{c}{**(3) Elution Gradient:**} \\ \hline  & & & & W—Washing step 100\% H\({}_{2}\)O (2 mL); & \\  & & & & A. & 100\% H\({}_{2}\)O (6 mL); & \\ • & Add 1 mL of H\({}_{2}\)O; & • & 3 mL CH\({}_{3}\)OH; & B. & CH\({}_{3}\)OH/H\({}_{2}\)O (50:50, 9 mL); & \\ • & Sonicate. & • & 6 mL H\({}_{2}\)O. & C. & CH\({}_{3}\)CN/H\({}_{2}\)O (70:30, 9 mL); & \\  & & & & D. & 100\% CH\({}_{3}\)CN (9 mL); & \\  & & & & E. & CH\({}_{2}\)Cl\({}_{3}\)/CH\({}_{3}\)OH (90:10, 9 mL). & \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Fractionation protocol of marine extracts on SPE/HR-X column (6 mL/500 mg) by using automated stepwise elution. Figure 1: Schematic outline of the fractionation process by the automated Solid Phase Extraction (SPE) procedure. More specifically, immunostimulatory activity was estimated as the ability to trigger response by immune cells [21, 22]. Specific activation was measured by release of interleukin-6 (IL-6) from human peripheral blood mononuclear cells (PBMCs). Activity was expressed as percentage of IL-6 induced by natural fractions in comparison with the maximal effect induced by PAM\({}_{3}\)CSK\({}_{4}\) (100% response), a synthetic triacylated lipopeptide that mimics the acylated amino terminus of bacterial lipoproteins and triggers the hetero-dimeric receptor composed of Toll like receptor (TLR) 1 and TLR2 [23]. Data analysis of the SPE protocol applied to evaluation of algal lipids as immunostimulants gave 44% positive hits over 27 tested samples (4% of the maximal production of IL-6). For eight of these samples, statistical analysis by Pearson's correlation between control (PAM\({}_{3}\)CSK\({}_{4}\)) and test groups identified a significantly increased activity with PBMC response after fractionation. It is worth noting that the high variability of the data is basically due the different response of the PBMC donors. Study on these extracts are currently in progress but, from a chemical point of view, it is to note that the activity was distributed among different fractions, thus suggesting the presence of different chemical effectors (Figure 3). In two cases (_Skeletonema marinoi_ FE65 and _Chaetoceros socialis_ FE17) we observed loss of activity after fractionation likely due to inherent chemical instability of the active components (Figure 3). Figure 2: Score hit over 47 marine samples tested for immunomodulatory and antimicrobial activity. (**a**) Response is reported as negative extracts (60%, pale blue), positive extracts (16%, pale green), positive extracts loosing activity after fractionation (4%, green) and positive hit only after fractionation (20%, dark green); (**b**) distribution of the activity within protists (\(n=34\)) (white) and sponges (\(n=13\)) (grey). Figure 3: Activity of microalgal samples (extracts and A–E fractions) tested for IL-6 production in human peripheral blood mononuclear cells (PBMCs). Activity is expressed as percent of maximal response induced by PAMMcSK4, a synthetic lipoprotein agonist of TLR1/2.