viverrini–associated Thai intrahepatic CCA. Antigens were retrieved from deparaffinized and rehydrated tissues by pretreating the slides in citrate buffer (pH 6.0) for 10 minutes at 108°C by way of autoclave. Immunohistochemical staining was performed using purified anti–MTA-1 immunoglobulin prepared as described.24, 29 Scoring was assessed semiquantitatively as negative (no detectable staining or positive staining in <10% of tumor cells); weakly positive (positive staining in 10%-25% of Selumetinib tumor cells); positive (positive staining in 25%-75% of tumor cells), and strongly positive (>75%) by two independent investigators. Quantitative real-time polymerase

chain reaction (PCR) was performed as described.24, 25-27 Sequences of primers are available on request. Differences among groups were compared using analysis of variance and the Student t test. P ≤ 0.05 was considered statistically significant. To investigate the influence of MTA1 on infection

and the establishment of O. viverrini, we isolated liver, small intestine, and kidney tissues from infected age-matched Mta1+/+ and Mta1−/− mice. Histopathological analyses using thin hematoxylin and eosin–stained sections revealed significant changes in the inflammatory response in Mta1+/+ and age-matched Mta1−/− mice. In particular, there was a higher occurrence of periductal fibrosis and infiltrating polymorphonuclear cells in the livers of wild-type mice compared with Mta1−/− mice (Fig. 1A; top panel). An increase in inflammatory response also correlated with a higher percentage (12%) of inflammatory zones in the Mta1+/+ mice. In addition, analysis KU-57788 mouse of hematoxylin and eosin–stained sections of the kidney supported the observation MCE公司 that O. viverrini infection resulted in a higher magnitude of inflammatory response in Mta1+/+ mice when compared with age-matched Mta1−/− mice (Fig. 1A, bottom panel).

To determine whether the presence or absence of MTA1 had a significant effect on the pathology associated with infection, levels of critical cellular markers known to be up-regulated during O. viverrini infection were evaluated using immunohistochemistry and quantitative reverse-transcription PCR (RT-PCR). We tested expression levels of CK-19, CK-18, and annexin-2. Expression of CK-19 has been widely used to study proliferation of biliary epithelium after O. viverrini infection, whereas annexin 2 appears to be a prognostic marker of O. viverrini infection-induced CCA.19, 31 There were significant increases in expression levels of CK-19, CK-18, and annexin 2, in the liver tissues from the Mta1+/+ mice when compared with age-matched Mta1 −/− mice using both immunohistochemistry (Fig. 2A-D) and quantitative real-time PCR (Fig. 2E-G). The T cell repertoire and secreted cytokines play an important role in determining the outcome of parasitic infections.