The average number of SFC in the absence of antigen was fewer than 10 (data not shown). Immediately after killing, liver was harvested, cut into small fragments and fixed in 10% buffered formalin, embedded in paraffin,

and cut into 5-µm sections. Liver sections were deparaffinized, stained with haematoxylin and eosin and evaluated under light microscopy by a ‘blinded’ qualified pathologist; the degree of liver inflammation, portal inflammation, bile duct damage, parenchymal inflammation and granuloma was scored as described previously [20–22]. Briefly, each section (except for those that showed bile duct damage or granuloma) was scored as either 0 = no significant change, 1 = minimal, 2 = mild, 3 = moderate or 4 = severe pathology. The sections that showed bile duct damage

and granuloma were scored as either https://www.selleckchem.com/products/midostaurin-pkc412.html 0 = no significant observation, 1 = low frequency Selleckchem EPZ6438 observed or 2 = frequently observed. All experiments were performed in triplicate and the data points shown are means of these triplicate analyses. The data are expressed as mean ± standard deviation (s.d.), and the significant differences between samples was determined using Student’s t-test. All analyses were two-tailed and P-values < 0·05 were considered significant. Statistical analyses were performed using Intercooled Stata 8·0 (Stata Corp, College Station, TX, USA). To evaluate the role of NK and NK T cells, we depleted NK and NK T cells by administering NK1·1 antibody. This treatment was confirmed to be effective due to the marked reduction in the frequency of NK1·1-positive NK cells or NK T cells by Stanford flow cytometry (Fig. 1). At both 6 and 12 weeks post-immunization, serum AMA were decreased significantly in

the NK1·1-depleted mice immunized with 2OA-BSA (n = 8) compared to sera from control mice immunized with 2OA-BSA. Interestingly, however, after 18 weeks there was no significant Bay 11-7085 difference in AMA titres in the two groups of animals (Fig. 2). As expected, there were no detectable AMA in BSA control mice. We evaluated T cell responses to PDC-E2 at 6, 12, 18 and 24 weeks using our ELISPOT assay in individual NK1·1-depleted and control 2OA-BSA immunized mice (Fig. 3). As noted, the numbers of IFN-γ-secreting T cells from the control 2OA-BSA-immunized mice both at 6 and 12 weeks were significantly higher than the 2OA-BSA-immunized NK1·1-depleted group. However, the mean number of such IFN-γ-secreting T cells was similar in both groups at 18 and 24 weeks. The coded series of liver tissues from the various groups of mice were studied by a pathologist blinded to the groupings of the donor mice. As seen in Fig. 4, there were no major differences in the degree of lymphoid cell infiltration in tissues from mice treated with the NK1·1 antibody compared with tissues from the control mice at 24 weeks. Both the levels of bile duct lesions and lymphoid cell infiltration appear to be mild in the NK1·1-depleted and control mice.