Biliary tract infection with the Group I carcinogenic liver fluke Opisthorchis viverrini is associated with severe inflammation leading to cholangiocarcinoma – a major biliary cancer in Southeast Asia. However, mechanism(s) by which the liver fluke induces host mucosal immune/inflammatory responses are unclear. In the present study we address whether a normal immortalized human cholangiocyte cell line (H69 cells) recognizes and responds to O. viverrini excretory/secretory products (OVES). Expression of multiple TLRs, activation of NF-κB, and expression of proinflammatory cytokines were monitored in the presence and absence of OVES. Our results showed that OVES induced increased cholangiocyte TLR4 mRNA expression, induced IκB-α degradation in a MyD88-dependent manner, and activated NF-κB nuclear translocation. Moreover, OVES induced expression and secretion of the strong chemoattractant chemokine interleukin 8 (IL-8) and pro-inflammatory cytokine IL-6. These results demonstrate that secreted/excreted products of O. viverrini are recognized by human cholangiocytes and initiate innate mucosal immunity/inflammatory cascades, a primary event in the pathogenesis of opisthorchiasis and cholangiocarcinoma.

BackgroundThe rhizome of Hydnophytum formicarum Jack., a medicinal plant known in Thai as Hua-Roi-Roo, has been used in Thai traditional herbal medicine for treatment of cancer. We assessed the ability of its ethanolic and phenolic-rich extracts and its major phenolic compound, sinapinic acid, possessing histone deacetylase (HDAC) inhibitory activity to inhibit proliferation of 5 human cancer cell lines.MethodsHeLa cells were used to study HDAC inhibitory activity of the extracts, sinapinic acid, and a well-known HDAC inhibitor sodium butyrate. Five human cancer cell lines and one non-cancer cell line were used to study antiproliferative activities of the plant extracts, sinapinic acid and sodium butyrate, comparatively.ResultsResults indicated that ethanolic and phenolic-rich extracts of H. formicarum Jack. rhizome possessed both antiproliferative activity and HDAC inhibitory activity in HeLa cells. Sinapinic acid, despite its lower HDAC inhibitory activity than the well-known HDAC inhibitor sodium butyrate, inhibited the growth of HeLa and HT29 cells more effectively than sodium butyrate. However, sinapinic acid inhibited the growth of HCT116 and Jurkat cells less effectively than sodium butyrate. The non-cancer cell line (Vero cells) and breast cancer cell line (MCF-7 cells) appeared to be resistant to both sinapinic acid and sodium butyrate. The growth inhibitory effects of the ethanolic and phenolic-rich extracts and sinapinic acid in HeLa cells were mediated by induction of apoptosis.ConclusionsThe results of this study support the efficacy of H. formicarum Jack. rhizome ethanolic and phenolic-rich extracts for the treatment of cervical cancer, colon cancer, and T- cell leukemia in an alternative medicine. Further studies of other active ingredients from this plant are needed.

Opisthorchis viverrini infection induces chronic inflammation, and a minor proportion of infected individuals develop advanced periductal fibrosis (APF) and cholangiocarcinoma (CCA). Inflammatory cytokines and/or their gene polymorphisms may link to these biliary pathologies. We therefore investigated associations among cytokine gene polymorphisms and cytokine production in 510 Thai cases infected with O. viverrini who presented with APF+ or APF−, as established by abdominal ultrasonography as well as in patients diagnosed with CCA. Levels of pro-inflammatory and anti-inflammatory cytokines were determined in culture supernatants after stimulation of peripheral blood mononuclear cells (PBMCs) with O. viverrini excretory-secretory (ES) products. Pro-inflammatory cytokines, IL-1β, IL-6, IFN-γ, LT-α, and TNF-α were significantly increased in CCA patients compared with non-CCA (APF− and APF+) cases. Polymorphisms in genes encoding IL-1β-511C/T, IL-6-174G/C, IFN-γ +874T/A, LT-α +252A/G, and TNF-α −308G/A were then investigated by using PCR-RFLP or allele specific-PCR (AS-PCR) analyses. In the CCA cases, LT-α +252A/G and TNF-α −308G/A heterozygous and homozygous variants showed significantly higher levels of these cytokines than the wild type. By contrast, levels of cytokines in wild type of IFN-γ +874T/A were significantly higher than the variants in CCA cases. IFN-γ +874T/A polymorphisms were associated with advanced periductal fibrosis, whereas IL-6 −174G/C polymorphisms were associated with CCA. To our knowledge, these findings provide the first demonstration that O. viverrini infected individuals carrying several specific cytokine gene polymorphisms are susceptible to develop fibrosis and CCA.

The carcinogenic liver fluke Opisthorchis viverrini causes chronic inflammation in the bile duct and resulting in unremitting tissue damage that lead to hepatobiliary diseases, including cholangiocarcinoma (CCA). Despite inflammatory cytokine expression having been studied in the animal model, so far no studies have been carried out on cytokines in human CCA cases. Here we report the profile of cytokine production in peripheral blood mononuclear cells (PBMCs) collected from O. viverrini-associated human CCA and uninfected normal controls after stimulation with O. viverrini excretory-secretory (ES) product. Eleven cytokine profiles including IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, TNF-α and LT-α measured by flow cytometry revealed both pro-inflammatory and anti-inflammatory cytokines were increased in the O. viverrini-associated CCA compared to uninfected normal controls. Specifically, net production levels of IFN-γ, IL-10, and LT-α were 40 to > 320 times higher in CCA than those of controls. These results suggest dysregulation of the immune response in the liver fluke associated CCA.

Opisthorchis viverrini is a carcinogenic parasite that can causes a bile duct cancercholangiocarcinoma. Study of immune response to this parasite in susceptible and nonsusceptible hosts may provide a clue to develop vaccines and immunodiagnostic markers, which are currently not available. Here, we compared the antibody response in susceptible Golden Syrian hamster and non-susceptible BALB/c mice infected by the liver fluke. In mice, the antibody was detected between one to two weeks post-infection, whereas it was positive between two to four weeks post-infection in hamsters. Immunolocalization revealed that the antibody from mice react strongly to the tegumental surface and gut epithelium of the worm, while hamster antibody showed a weak signal in the tegument and a comparable signal in the gut of the worm. Immunoblot of the tegumental proteins demonstrated that while hamster antibody showed a broad specificity, mice strongly reacted to a single protein band.Mass spectrometry revealed these immunogenic targets. Recombinant proteins of the reactive targets were produced in the bacterial expression system. The immunoblot of these recombinant proteins confirm the reactivity of their native form. In summary, there is a different antibody response against O. viverrini infection in susceptible and non-susceptible hosts. The non-susceptible host react quicker and stronger than the susceptible host.