?(Fig.6B).6B). CT. LT-IIb significantly enhanced the number of AgI/II-specific antibody-secreting cells in the draining superficial cervical lymph nodes compared to LT-IIa and CT. LT-IIb and CT induced significantly higher plasma anti-AgI/II IgG titers compared to LT-IIa. When LT-IIb was used as adjuvant, the proportion of plasma IgG2a relative to IgG1 anti-AgI/II antibody was elevated in contrast to the predominance of IgG1 antibodies advertised by AgI/II only or when CT or LT-IIa was used. In vitro activation of AgI/II-specific cells from your superficial lymph nodes and spleen exposed that LT-IIa and LT-IIb induced secretion of interleukin-4 and significantly higher levels of gamma interferon compared to CT. These results demonstrate that the type II HLT LT-IIa and LT-IIb show potent and unique adjuvant properties for revitalizing immune reactions to a noncoupled protein immunogen after mucosal immunization. The heat-labile enterotoxins (HLT) of and constitute a family of bacterial toxins that are related in structure and function (10, 11, 16, 35). Both are oligomeric protein toxins composed of one A polypeptide and five B polypeptides in which the quaternary structure is managed by noncovalent bonds between the A polypeptide and a pentameric ring of B subunits (7, 13, 32). The biological effects of the enterotoxins are determined by the binding specificity of the fully put together B subunits and the enzymatic activity of the A subunit. The pentameric ring formed from the B subunits mediates binding to the sugars residues of gangliosides present on the surface of various eukaryotic cells (3, 18). Two serogroups of HLT have been distinguished on the basis of unique immunoreactivity (15, 28). Serogroup I consists of cholera toxin (CT) and the HLT LT-I, which includes two antigenic variants isolated from humans and pigs, designated LTh-I and LTp-I, respectively (19, 28). Serogroup II enterotoxins include type II HLT in the beginning Brucine designated LT-like toxins and later called LT-II enterotoxins (9). Based on immunoreactivity and amino acid sequence homology, two antigenic variants of LT-II, designated LT-IIa and LT-IIb, have been isolated (9C11, 17). Although serogroup I and serogroup II enterotoxins induce related morphological effects on Y1 adrenal Mmp10 cells and activate adenylate cyclase in cell ethnicities, both LT-IIa and LT-IIb look like more potent than either CT or LT-I in Y1 adrenal cell Brucine assays; however, neither LT-IIa nor LT-IIb induces the typical fluid build up in ligated ileal loops observed with CT and LT-I (16). In human being T84 intestinal cells, only CT elicited a cyclic AMP-dependent chloride response that is responsible for the massive effusion of water into the lumen of the gut (39). Assessment of the expected amino acid sequences of type I and type II enterotoxins discloses a large degree of variability. While the B polypeptides of CT and LT-I show over 80% homology to each other, both CT and LT-I have less than 14% amino acid sequence homology to the B subunits of either LT-IIa or LT-IIb (15, 28C30). The considerable diversity in amino acid sequences between type I and type II HLT not only results in antigenically distinct organizations but also imparts different ganglioside binding specificity to the respective B subunits. Specifically, the high-affinity receptor for CT and LT-I offers been shown to become the monosialoganglioside GM1, while the B subunit of LT-IIa binds with high affinity to GD1b and less strongly to GM1, GT1b, GQ1b, GD2, GD1a, and GM2 (6). Unlike CT and LT-IIa, LT-IIb lacks affinity for GM1 but offers been shown to bind with high affinity Brucine to the disialoganglioside GD1a (6). Gangliosides are sialic acid-containing ceramide oligosaccharides in which the polar head groups consist of carbohydrate moieties having a lipophilic ceramide tail anchored in the lipid.